NCT showed cytotoxic effects against both strains of T. vaginalis
tested. Treatment with 55 mM NCT (1%), a concentration well tolerated by all human tissues tested so far 
, led to 100% killing of both strains tested within 15 min in TYM medium (). At lower concentrations in this medium, strain ATCC 30001 was more susceptible than strain ATCC 50138. The EC50
of NCT was 9.0 mM for strain ATCC 30001, whilst it was 28.7 mM for strain ATCC 50138 after 1 h of treatment. EC90
values were 9.8 mM and 36.7 mM, respectively.
Fig. 1 Treatment of Trichomonas vaginalis ATCC 30001 with N-chlorotaurine (NCT) and co-treatment with NH4Cl in trypticase–yeast extract–maltose (TYM) medium: , 55 mM (1%) NCT; , 10 mM (0.18%) NCT; ○, (more ...)
The activity of NCT was markedly enhanced by the addition of NH4
Cl, explained by formation of the small and more lipophilic monochloramine (NH2
Cl), which penetrates pathogens better than NCT and leads to faster oxidation of intracellular proteins [6–8]
. In strain ATCC 30001, only one-third of the concentration of NCT was needed to achieve 100% killing when NH4
Cl was added, i.e. 17 mM (0.3%) NCT plus 57 mM (0.3%) NH4
Cl (); moreover, the killing time was reduced to 5 min. This effect was even greater when experiments were carried out in PBS instead of culture medium. In PBS, co-treatment with 5.5 mM (0.1%) NCT plus 19 mM (0.1%) NH4
Cl also resulted in complete killing of trichomonads within 5 min (data not shown). Inactivation of NCT and the formed NH2
Cl by reaction with constituents of the TYM medium must be assumed to contribute significantly to delayed killing in this medium, particularly at concentrations below 10 mM. Such chlorine consumption in the presence of organic material, in particular reducing thio compounds, is well known [6,7]
. It may also explain the relatively sharp threshold between highly active and inactive concentrations of NCT (). In strain ATCC 50138, higher concentrations of NH4
Cl (molar ratio 1:29) were necessary to achieve enhancement of activity in TYM medium.
The metronidazole-sensitive strain (ATCC 30001) was more susceptible to NCT than the metronidazole-resistant strain (ATCC 50138), and this difference was statistically significant (P
< 0.001). An approximately three- to four-fold concentration of NCT required to kill 50% of the metronidazole-sensitive trichomonads was required to achieve the same killing rate in the metronidazole-resistant strain. Although the modes of action are different, since both metronidazole and NCT produce oxidative stress one could hypothesise that the more resistant strain has a better anti-oxidative protection system. This may be the case, but at the applied millimolar concentrations of NCT such mechanisms would not be sufficient. Since penetration of the pathogen rather than oxidation of the surface is the decisive step for killing by NCT 
, we assume that several factors, particularly differences in the membrane composition, may play a major role as has already been observed in other microorganisms 
In vivo studies have shown that 55 mM NCT (1%) is well tolerated by human mucosal tissue 
. The in vitro tolerability of human cells in cell culture to NCT is known to be significantly (ca. 50–100-fold) lower [13–15]
. However, vaginal epithelial cells were observed to be relatively resistant to NCT in the current study. The EC50
of NCT for these cells in vitro was 5.5 mM and the survival rate was 25% after treatment with 55 mM NCT for 1 h (). Tolerability to NCT was altogether significantly higher than to metronidazole, the standard drug for treatment of trichomoniasis. Increased tolerability of human cells to the agent in vivo compared with that in vitro can be explained by the fact that within tissues the cells are coherently organised in a structure affording better protection to oxidative agents. This has already been proven with human skin in vivo as well as human epidermis carcinoma cells in vitro 
Survival of vaginal epithelial cells treated for 1 h with N-chlorotaurine (NCT) (full diamond) or metronidazole (open square).
The stability of the aqueous solution of NCT depends on temperature. At 37 °C its oxidation capacity decreases to zero within 3 weeks, whereas it remains at 75% after storage at room temperature for 2 months 
. At sites of inflammation, oxidation capacity decreases more quickly owing to halogen-consuming proteins. After 15 min the oxidation capacity is reduced by 30–50% and after 2 h by up to 90% 
. However, as trichomonads are killed within 15 min, and even faster with the addition of NH4
Cl, these decomposition rates are not likely to affect a possible therapeutic approach against trichomoniasis. Moreover, since human exudates demonstrated enhanced rather than decreased microbicidal activity of 0.5–1% NCT after topical application [8,9]
, it may be expected that NCT is also microbicidal against T. vaginalis
in vivo after topical application.
In conclusion, we have shown that NCT has strong trichomonacidal effects that are further enhanced by the addition of NH4Cl. Further investigations of its utility in T. vaginalis infections are required.