The evaluation of the effect of latex condoms using cell culture techniques

Abstract

Increased awareness of protection against infections such as Hepatitis Band Human Immune-deficiency Virus (HIV)/ Acquired Immune Deficiency Syndrome (AIDS) and other sexually transmitted diseases has led to an increase in the demand for latex gloves and condoms leading to an increase in latex allergy. Besides latex, condoms also contain several undisclosed chemicals including antioxidants, accelerators, emulsifiers, stabilizers, lubricants, and in some cases flavourings and colourants. Though extensive testing is done to evaluate the physical quality of condoms, little information is available regarding the biological safety of condoms. In this study a modification of the direct cell culture testing method that is specified by the American Test Method F813-83 of 1998 was used to determine the cytotoxicity of the surface material of latex condoms prepared at time intervals that represents normal physiological exposure times T2, T4 and T8. The L929 cells were exposed to medium containing increasing amounts of condom washings (0-66%) for 20 hours. After exposure cell number and viability was determined using the Crystal violet (CV) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-3H-tetrazolium bromide (MTI) assays respectively. Data was evaluated using a split-plot design with the appropriate Analysis of Variance (ANOVA). The effect of the condom washings on cell morphology and CV staining, MTI metabolism and Neutral red (NR) uptake at a fixed condom washing (16%) and exposure time T8 was evaluated microscopically. Cell membrane integrity was evaluated by Propidium iodide (PI) uptake and with PI staining after fixation and Hoechst 33324 (H33342) staining nuclear structure was evaluated with fluorescence microscopy. Apoptosis induced DNA fragmentation was evaluated by agarose gel electrophoresis. The effects of condom washings at 16% condom washing and exposure times T2, T4 and T8 was further evaluated in the HeLa cell line, a cell line in origin and type closer to that of the cervical lining. Cytotoxicity was evaluated using the CV, MTT and NR assays. In the L929 cell line, condom types Non-lubricated condoms (NLC), Lubricated condoms (LC) and Lubricated and flavoured condoms (LFC) behaved differently over time of exposure and the concentrations of condom washings. LFC were found to induce a decrease in cell number compared to other condom types, followed by LC and NLC revealed increases in cell number. Split-plot analysis, revealed that condom type x time (CT x Time) is significantly different due to the effect observed at T2 for LC. The MTT usually considered being more sensitive than the CV assay showed only toxicity for LFC and not for NLC and LC as with the CV assay. Exposure to LFC revealed significant decrease of 70 % decrease in cell viability at T8. Condom washings, LC, LFC and LFCC had no effect on cell morphology following CV staining. MTT metabolism and NR uptake was reduced and altered cell morphology was observed for L929 cells exposed to LFC and LFCC. Little PI uptake was observed for all cells exposed to condom washings. Condensed nuclei were observed for L929 cells exposed to LFC and LFCC while Hoechst staining revealed peripheral arrangement of DNA with Hoechst 33342 staining. Cell death in L929 cells were found to be mediated by apoptosis with L929 exposed to LFC showing the most damage. All effects of LFC is greater than that observed for LFCC indicating that other factors rather than the number of components present in each type of condom may account for toxicity. Toxicity of condom washings were compared to that found in the L929 cell line using the CV and MTT assays and an additional bioassay the NR assay was included. Condom types, LC, LFC and LFCC had a significant effect on cell viability and lysosomal membrane integrity. Differences observed between the L929 and HeLa cells were due to the increased viability observed for LC and the decrease in membrane integrity for LFC on HeLa cells. With LC and LFC no decrease in cell number and viability was observed as previously reported for the L929 cell line. Although no decreased in cell viability is observed for LFC a decrease of 75% in lysosomal membrane integrity is observed. The increase in cell viability found for HeLa exposed to LC (although statistically not significant) cannot be explained. Changes in cell viability and membrane integrity was only observed for HeLa cells, indicating that the HeLa cell line is more sensitive to the cytotoxic effects of condom washings. Furthermore the NR assay is a more sensitive assay than the MTT assay in detecting the cytotoxic effects of LFC condom washings at low concentrations. These are assays address only the effects of short-term exposure and not possible genotoxic effects that may occur following repeated and long-term exposure as reported in other latex products.