SUDOL, KRISTIN M. BA; PHILLIPS, DAVID M. PhD
From the Population Council, New York, New York
This work was supported by The United States Agency for International Development (USAID). The authors thank Malka Kohn, Kanani Titchen, Gianina Aronovici, Mitchell Thorn, Robin Maguire, and Andrea Wallace for their help, advice, and encouragement.
Correspondence: David M. Phillips, PhD, Population Council, 1230 York Avenue, New York, NY 10021. E-mail: email@example.com
Received for publication October 15, 2003, revised January 14, 2004, and accepted January 15, 2004.
Objective: The current study was conducted to determine the relative safety of sexual lubricants for rectal use.
Goal: Our goal was to identify a sexual lubricant that would not damage the rectal epithelium.
Study Design: We describe a mouse assay to determine the degree to which lubricants cause rectal sloughing. We also tested lubricants for cytotoxicity and their effect on Herpes simplex virus (HSV-2) infection after rectal challenge.
Results: Of the products tested, Delube and KY-Plus are most likely to damage the rectal epithelium, whereas Viamor, Vagisil, and Astroglide would cause some degree of rectal damage. PBS, Carraguard, and methylcellulose were not toxic in any of the assays.
Conclusion: We have developed a quantitative method to assay the degree of sloughing of the rectal epithelium. Using this and other techniques, we are able to predict the degree to which lubricants could be safe for rectal use.
THE LARGE MAJORITY OF men who have sex with men (MSM) use lubricants during rectal sex. 1 Several years ago, a large survey of MSM found that 42% of the men reported that they chose lubricants containing N-9. 1 However, we have demonstrated that rectal application of N-9 in mice enhances the probability that an animal will become infected after rectal challenge with Herpes simplex virus (HSV-2). 2 Furthermore, studies have demonstrated that N-9 causes rapid sloughing of the rectal epithelium in animals and humans. 2-4 These observations lead a number of organizations, including the Centers for Disease Control and Prevention (CDC), 5 the World Health Organization (WHO), and CONRAD, 6 to recommend that N-9 products should not be used rectally.
These findings raise the question of which sexual lubricants are safest for rectal use. Many sexual lubricants contain high concentrations of cytotoxic preservatives and/or excipients that could possibly result in damage to the rectal epithelium (similar to that caused by N-9).
This report describes a direct and nonlabor-intensive mouse model that can be used to determine the degree to which a lubricant causes sloughing of the rectal epithelium. In addition, a standard cytotoxicity assay has been used. Enhancement of HSV-2 infection after rectal challenge was used to assay the degree to which lubricants enhance infection. Using these methodologies, several lubricants could be compared in terms of rectal safety.
Materials and Methods
Test formulations were water-based Vagisil (Combe Inc., White Plains, NY), Astroglide (Biofilm Inc., Vista, CA), Viamor (Women First HealthCare Inc., San Diego, CA), and Delube (E-Gal Corp., Highland Park, IL), carrageenan-based Carraguard-an investigational new drug of the Population Council 7 -and a methylcellulose formulation. 7 KY-Plus (containing nonoxynol-9; Advanced Care Products, Raritan, NJ) was used as a positive control and phosphate-buffered saline (PBS) (Gidco, Grand Island, NY) served as a negative control.
Six- to 8-week-old female BALB/c mice (Charles River, Wilmington, MA) were maintained on a 12-hour light cycle at the Rockefeller University Laboratory Animal Research Center according to university guidelines. Animals were group housed, and food and water was available ad libitum.
Methods for propagating and titering HSV-2 have been previously described. 8
MTT Cytotoxicity Assay
Cytotoxicity of serial dilutions of compound was assayed using the MTT (thiazolyl blue) colorimetric assay, in which color saturation (absorbance) is directly proportional to cell density. Propagation of ME-180 epithelial cells has been previously described with the following changes. 9 Fifty microliters of RPMI (Invitrogen, Carlsbad, CA) containing various concentrations of the test compound dilutions were added to a Falcon 96-well assay plate (Becton Dickinson, Franklin Lakes, NJ) containing confluent epithelial-like human cervical (ME-180) cells. Plates were incubated for 2 hours at 37°C and 5% CO2. Wells were subsequently washed 3 times with 100 μL of RPMI. Twenty microliters of MTT reagent (Promega, Madison, WI) was added to each well. After a 1-hour incubation period at room temperature, absorbance was read on a plate reader at 490 nm.
Rectal HSV-2 Challenge
Food was withheld for 24 hours. Subsequently, 0.1 mL of a solution of 1 mL of dissolved Ketamine (Fort Dodge Laboratories, Fort Dodge, IA) with 0.5 mL of xylazine (Miles Inc., Shawnee Mission, KS) in 6.75 mL PBS was injected intraperitoneally (IP) to immobilize the animals and prevent defecation. Once anesthetized, mice were rectally treated with 20 μL of test formulations using a positive placement Gilson Microman pipette. Five minutes later, mice were challenged rectally with 10 μL of DMEM (Dulbecco's Modified Eagle Medium; Gibco, Bethesda, MD) containing 5 × 104 plaque-forming units (PFU; 50% infection dose) of HSV-2 using a M20 Pipetman. A 14-day observation period followed rectal challenge. Animals displaying redness, swelling, hair loss, and/or lesions in the rectal area were scored as positive and euthanized.
It has been previously observed that N-9 causes rectal sloughing in mice, monkeys, and humans. 2-4 Based on this observation, an assay to quantify the number of epithelial cells in the rectum following product insertion was developed. To carry out these studies, food was withheld and animals were anesthetized as described previously. Once anesthetized, 20 μL of test formulation was placed in the mouse rectum using a Microman. Fifteen minutes later, the rectum was lavaged 5 times with 20 μL PBS. The 5 lavages were pooled, and 10 μL trypsin-EDTA was added. Subsequently, the washes were incubated at 37°C and 5% CO2 for 15 minutes. This procedure was carried out to separate individual epithelial cells from each other. After incubation, 10 μL of fetal bovine serum (Invitrogen, Carlsbad, CA) was added to inhibit trypsin. Cells were counted in a Coulter Z2 Particle Count (Coulter Corp., Miami, FL). Each sample was counted 3 times. The averages from each sample reading were calculated, and the standard deviation was determined.
Results are expressed as mean ± standard deviation (SD). Data were subjected to analysis of variance for group analysis of variance. A P value ≤0.05 was considered significant.
MTT Cytotoxicity Assay
The degree of cytotoxic effect varied widely. Carraguard and methylcellulose were not cytotoxic, even when diluted 1:1 with medium. KY-Plus was 50% cytotoxic as compared with control (no formulation) when diluted 330 times (Fig. 1;Table 1).
Infection Assay Because we wanted to determine protection from or enhancement of infection, we chose a dose of HSV-2 known to infect 50% of the mice. Carraguard showed significant protection from infection (P <0.05) as compared with PBS. Delube and KY-Plus significantly increased infection as compared with PBS (P <0.01) (Fig. 2;Table 2).
Rectal Sloughing Assay
The average number of cells that were sloughed varied widely among products. The number of cells recovered after treatment of methylcellulose and Carraguard formulations was similar (approximately 2 × 106/mL). In sharp contrast, more than 10 times the number of cells (approximately 6 × 107/mL) was recovered from mice that were treated rectally with Viamor, Vagisil, or Astroglide. All were highly significant (P <0.01) compared with PBS. At least another order of magnitude of cells (approximately 8 × 108/mL) was recovered from the rectums of mice pretreated with Delube or KY-plus (Fig. 3). This difference was highly significant compared with Viamor, Vagisil, or Astroglide (P <0.01).
Three different assays were used to determine the relative deleterious effect on the rectal epithelium of several sexual lubricants. The findings are summarized in Table 2. As shown by our assays, 1 of these products, Delube, which contains benzalkonium chloride, is just as cytotoxic for rectal use as KY-Plus, a 2.2% N-9 product. The other sexual lubricants tested, Astroglide, Vagisil, and Viamor, are far less cytotoxic than Delube, but they still have significant rectal cytotoxicity. The only products that do not exhibit any cytotoxicity are Carraguard and methylcellulose, which are not yet commercially available. We plan to use the assays described here to test more commercially available sexual lubricants in the hope of identifying those that appear to be safe for rectal use.
Recently, Baron et al. 10 assayed several sexual lubricants for activity against HIV in vitro. They found that some sexual lubricants, including Astroglide, Vagisil, Viamor, and N-9, blocked HIV infection of lymphoma cells. The authors suggested that these products should be explored as possible HIV preventatives. We caution that any anti-HIV properties are most likely related to general cytotoxicity. The formulations with the greatest anti-HIV activity are likely to breach the delicate epithelia of the rectum and lower intestinal tract. Thus, although they could have some anti-HIV activity, these products should not be used as rectal lubricants.
Preservatives and/or excipients contained in the lubricants could be the agents responsible for rectal cytotoxicity. In this regard, both Carraguard and the methylcellulose formulation have sufficient concentrations of preservatives to pass the preservative effectiveness tests required by the U.S. Food and Drug Administration for vaginal products, and they contain no other excipients. Thus, we speculate that the concentration or type of preservatives and/or excipients in some sexual lubricants could be excessive.
We have developed a sensitive quantitative assay to measure cell sloughing of the rectal epithelium. Although this assay measures rectal epithelial sloughing in mice only, human rectal studies have been found to correlate strongly to murine rectal studies. 3 Other current animal systems that access topical cytotoxicity are problematic. The rabbit eye model is not sensitive enough to distinguish among products with relatively minor differences in topical cytotoxicity. 11 Similarly, in the rabbit dermal irritation study, minor differences in irritability are difficult to detect. 11 Although the rabbit vaginal irritation model is quantitative, it is somewhat subjective because it relies on relative scores from examination of histology. 11,12 In addition, these assays are costly, because rabbits are relatively expensive animals. The system described here is quantitative, is not very time-consuming (cells are counted in a Coulter counter), and is inexpensive because mice are relatively inexpensive. Thus, the cell sloughing assay described here could be useful for assaying topical cytotoxicity of formulations under development for a variety of topical applications.
Lastly, we emphasize that this study deals exclusively with rectal safety. The rectal epithelium is an absorptive tissue with a delicate simple columnar epithelium. In sharp contrast, the human vagina is lined by a multilayered stratified squamous epithelium designed for the strain that can occur during sexual intercourse and childbirth. Thus, products that could be hazardous for rectal use could be safe for vaginal use. For instance, vaginal spermicides containing N-9 are of particular importance because, in many cases, they are the only contraceptives readily available to certain populations of women. No conclusions can be drawn about vaginal safety of sexual lubricants from this article.
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2. Phillips DM, Zacharopoulos VR. Nonoxynol-9 enhances rectal infection by herpes simplex virus in mice. Contraception 1998; 57:341-348.
3. Phillips DM, Taylor CL, Zacharopoulos VR, Maguire RA. Nonoxynol-9 causes rapid exfoliation of sheets of rectal epithelium. Contraception 2000; 62:149-154.
4. Patton DL, Cosgrove Sweeney YT, Rabe LK, Hillier SL. Rectal applications of nonoxynol-9 cause tissue disruption in a monkey model. Sex Transm Dis 2002; 29:581-587.
5. Centers for Disease Control and Prevention. Sexually Transmitted Disease Treatment Guidelines 2002. MMWR 2002; 51(No. RR-6):3-5.
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7. Maguire RA, Zacharopoulos VR, Phillips DM. Carrageenan-N9 spermicides for preventing pregnancy and sexually transmitted infections. Sex Transm Dis 1998; 25:494-500.
8. Zacharopoulos VR, Phillips DM. Vaginal formulations of carrageenan protect mice from herpes simplex virus infection. Clin Diagn Lab Immunol 1997; 4:465-468.
9. Tan X, Pearce-Pratt R, Phillips DM. Productive infection of a cervical epithelial cell line with human immunodeficiency virus: Implications for sexual transmission. J Virol 1993; 67:6447-6452.
10. Baron S, Poast J, Nguyen D, Cloyd MW. Practical prevention of vaginal and rectal transmission of HIV by adapting the oral defense: Use of commercial lubricants. AIDS Res Hum Retroviruses 2001; 17:997-1002.
11. Gad SC, Chengelis CP. Acute Toxicology Testing. San Diego: Academic Press, 1998.
12. Association of Food, Drug Officials of the United States. Appraisal of the Safety of Chemicals in Foods Drugs and Cosmetics. Topeka, KS: Association of Food and Drug Officials of the United States, 1965.