Aspects of the effect of thioxanthone on Schistosoma mansoni in mice and in vitro.

Notes Aspects of the effect of thioxanthone on Schistosoma mansoni in mice and in vitro HARRY G. LE 1 The efficacy of the thioxanthone drugs, lucanthone and hycanthone, in the treatment of experimental and clinical Schistosoma mansoni infections appears to be well established. Until recently (Lee & Cheever, 1970; Foster et al., 1971) the numerous laboratory studies of these agents had produced few data concerning possible differential effects on male and female parasites. Hill (1956) briefly stated that female parasites preferentially survived subcurative lucanthone treatment in mice, but most investiga- tions into thioxanthone chemotherapy either were not concerned with such an effect or did not find it. Another striking aspect of the published work on experimental thioxanthone chemotherapy is the vari- ation in dose-response relationships, even within a single host species, in different laboratories. The study described herein was designed to investigate the parasiticidal and sublethal effects of these thioxan- thone agents on both male and female S. mansoni in infected mice and in vitro. Materials and methods The NIH Puerto Rican (NIH-PR) strain of Schisto- soma mansoni was employed in all experiments. However, strains of different geographical origin were occasionally used for comparison with NIH-PR (Lee et al., 1971). Shedding of cercariae was gener- ally induced from pooled infected snails. When uni- sexual infections were desired, cercariae from single snails previously exposed to one miracidium (NIH- PR strain) were used. Female Swiss albino mice weighing 18-20 g were exposed to a measured num- ber of cercariae by tail-immersion using a modifica- tion of the technique of Olivier & Stirewalt (1952). Mice were later randomly assigned to control and treatment groups. Chemotherapy consisted of one of the following regimens: 5 daily doses of lucan- thone by gavage, 5 daily doses of stibophen intra- 1 Formerly: Research Associate, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md., USA. Present address: The George Williams Hooper Foundation, San Francisco Medical Center, University of California, USA. peritoneally, or, a single intramuscular dose of hycanthone base dissolved in castor oil. Except as noted in the results, treatment of mice was begun 48 days after exposure to cercariae, and mice were sacrificed and perfused by the method of Duvall & DeWitt (1967) 21-25 days later. The in vitro assays were performed with hycan- thone methanesulfonate (Sterling-Winthrop), which is water-soluble. The method of Lee & Michaels (1968) was employed with the exception that the culture medium generally lacked serum enrichment and the period of observation was extended to 7 days. Serial two-fold dilutions of drug were employed in each trial. Results The lethal effects of lucanthone on parasites of each sex in two selected groups of mice are shown in Table 1. The results of a similar study using hycanthone treatment are given in Table 2. The parasiticidal effects of hycanthone methanesulfonate in vitro are shown in Table 3. Tables 1 and 2 show that male worms were more susceptible to the lethal action of lucanthone and hycanthone than were females. Males ofthe St Lucia, Liberia, and Belo Horizonte (Brazil) strains were likewise more susceptible than females to both drugs. This difference was independent of the initial worm burden and was not a feature of the response to stibophen or niridazole. Entirely comparable results were obtained in an experiment in which mice were perfused 60 days after treatment rather than after the customary 21-25-day period. From several trials (NIH-PR strain) it appeared that 25 mg of hycan- thone per kg of body weight killed about 70% of male worms and that 50 mg/kg killed about 30% of females regardless of whether infections were uni- sexual or bisexual. Females surviving doses lethal for males were stunted and lacked gut pigment (Fig. 1). It was interesting to note that in mice treated with 50 mg of hycanthone per kg of body weight, female NIH-PR worms had returned to their 2815A - 397 NOTES Table 1. Examples representative of the parasiticidal effect of lucanthone (gavage daily for 5 days) by sex * ParasieDaily Mean no. of live worms per mouse Worm survival as Parasite dose (± SE) percentage of control No.strain g of mice(mg/kg) Male Female Male Female NIH-PR control 11.7 (0.8) NIH-PR 25 9.4 (1.2) NIH-PR 50 7.9 (1.5) NIH-PR 75 3.6 (1.2) NIH-PR 100 1.8 (0.6) 18.2 (1.4) 14.7 (1.6) 12.8 (1.6) 14.4 (1.4) 15.6 (1.3) 100 100 17 80 81 68 70 31 79 10 9 10 15 86 10 Liberia control 12.2 (1.5) Liberia 75 3.0 (0.8) Liberia 100 0.83 (0.17) Liberia 150 0.33 (0.17) Liberia 200 0.29 (0.18) 6.9 (0.7) 6.1 (1.1) 5.7 (1.0) 4.9 (1.1) 3.7 (0.4) 100 100 25 88 7 83 3 71 2 54 16 8 6 9 7 * The experiments represented here are the same as those for which results on the male worms have been previously reported (Lee et al., 1971). Table 2. Examples representative of the parasiticidal effect of hycanthone (single dose, intramuscularly) by sex * Parasite Daily Mean no. of live worms per mouse survival as trasine Doy (± SE) percentage of No. of(mg/kg)os o trol mice Male Female Male Female NIH-PR control NIH-PR 20 NIH-PR 25 NIH-PR 30 NIH-PR 40 NIH-PR 50 St. Lucia control St. Lucia 20 St. Lucia 25 St. Lucia 30 St. Lucia 40 St. Lucia 50 10.8 (1.4) 8.0 (0.90) 3.7 (1.1) 3.2 (0.73) 0.9 (0.18) 0.7 (0.35) 8.8 (0.69) 7.3 (1-3) 7.4 (1.0) 7.9 (1.2) 4.9 (0.94) 5.1 (1.1) 10.0 (0.83) 6.9 (0.71) 8.0 (0.75) 6.4 (0.76) 5.6 (0.65) 7.1 (0.64) 6.5 (0.69) 7.0 (1.0) 5.9 (1.0) 8.6 (1.2) 7.0 (0.97) 7.4 (1.0) 100 100 74 69 34 80 30 64 8 56 6 71 100 100 83 108 84 93 90 134 56 108 58 114 17 10 10 9 10 10 17 10 10 10 9 9 * The experiments represented here are the same as those for which results on the male worms have been previously reported (Lee et al., 1971). 398 Fig. 1. Stunted worms recovered from mice treated with 50 mg of hycanthone/kg body weight 58 days previously and not reinfected. Fig. 2. Worms that appear normal recovered from mice treated with 50 mg of hycanthone/kg body weight 58 days previously and reinfected with male cercariae 53 days previously. Most male worms in these mice are presumed to be from the challenge infection by male cercariae. The females, however, are equivalent to those shown in Fig. 1 except that new males were supplied by the second exposure. EFFECTS OF THIOXANTHONE ON SCHISTOSOMA MANSONI normal adult appearance by 4 months after a chal- lenge infection with male cercariae (Fig. 2), but there was no microscopical evidence of ova in host viscera or stools. In contrast, surviving NIH-PR male worms in mice given a dose of 25 mg/kg stimulated production of viable ova by 2 months after a chal- lenge infection with female cercariae. In vitro, hycanthone methanesulfonate proved to be highly active against S. mansoni (Table 3). At the higher effective drug concentrations, worms were stained yellow by the drug even while they were still actively motile. Death of worms was observed after an exposure of as little as 24 hours to the higher concentrations, and at lower drug levels most worms were dead after exposure for 7 days. How- ever, worms exposed to low drug concentrations occasionally died in the second week of incubation while control worms always survived for at least 2 weeks. Enrichment of the basic medium with fetal calf serum decreased the effectiveness of the drug. A remarkable and consistent feature of the drug's activity in vitro was the greater killing effect on male worms. Discussion Experimental. It appears that the thioxanthone drugs, lucanthone and hycanthone, exert a much Table 3. Effect of hycanthone methanesulfonate on worm survival in vitro. Minimum lethal Expo- concentration Trial Repli- Medium b sure (Mg/ml)cate a h Male Female 1 1 199 168 0.63 1.3 2 199 168 0.63 1.3 2 1 199 168 0.16 1.3 2 199 168 0.32 1.3 3 199 168 0.32 1.3 4 199 + 10% FCS 168 2.5 5.0 5 199+10%FCS 168 1.3 5.0 3 1 199 24 5 20 2 199 24 5 40 1 c 199 24 1.3 10 2 c 199 24 0.63 10 4 1 c 199 24 0.9 7.1 2 c 199 24 0.9 7.1 a One pair of worms at each drug concentration in each replicate. b Medium 199 was obtained from Grand Island Biological Co., Grand Island, N.Y., USA. FCS = fetal calf serum. c Read 6 days after transfer to drug-free medium. greater effect on male S. mansoni than on females and that the effectiveness of these agents on mouse infections is less than has been generally reported (Azim et al., 1948; Kikuth & G6nnert, 1948; Gonnert & Vogel, 1955; Berberian & Freele, 1964; Berberian et al., 1967). Of the many studies published, only the recent work of Foster et al. (1971) agrees qualita- tively and quantitatively with the present data. As pointed out by the above authors, the use of techniques other than portal perfusion to study drug effects seems the best explanation of the earlier failure to demonstrate that some or all of the female worms survived treatment. By the technique of Duvall & DeWitt (1967) these stunted depigmented female worms (Fig. 1) rarely, if ever, escape detec- tion. Hosts infected intraperitoneally (Berberian & Freele, 1964) must be perfused after rinsing away immature peritoneal worms (Moore & Meleney, 1955), which closely resemble the widowed females. It is not clear why the results of Thompson et al. (1962) with lucanthone did not indicate a preferen- tial survival of females. At least some of the males that survived a dose of 25 mg/kg were able to stimulate normal females to lay fertile ova. With the exception of one infec- tion, it appears that the female parasites that sur- vived in mice treated with 50 mg of hycanthone per kg of body weight, and that were supplied with male partners shortly thereafter, produced no eggs for 4 months after treatment. Apparently this loss of fertility may not be a permanent effect of this regimen, as parasite fertility in mice treated with hycanthone methanesulfonate has been destroyed temporarily only to reappear 3-12 months later (Rogers & Bueding, 1970) in association with inherit- able resistance to the drug on the part of the parasite (Rogers & Bueding, 1971). It is not known, at present, why female worms are less susceptible to the lethal action of the thioxanthone drugs. The foregoing results in vitro suggest that the differential susceptibility of males and females is innate and not host-mediated. It is interesting to note that the in vivo uptake of hycan- thone methanesulfonate is actually greater in female than in male worms (Yarinsky et al., 1970). Epidemiological implications Since the preferential effect of hycanthone on male S. mansoni cxists both in mice and in vitro, it seems appropriate to speculate in terms of natural hosts such as man. Patients treated with hycanthone gen- erally show a 90-100% decrease in egg passage, and 9 401 402 NOTES one would suspect persistence ofnumbers of unpaired female worms. While a degree of immunity to reinfection is gen- erally conceded to exist in populations with a high prevalence of Schistosoma mansoni infections, the stimulus to such immunity is not well defined. The significance of premunition in several parasitic infec- tions is widely appreciated. The "concomitant immunity " in the rhesus monkey against S. mansoni (Smithers, 1968) suggests that, perhaps in man as well, an established infection with adult worms may be the essential stimulus for immunity against re- infection. Since infections with worms ofone sex are expected to be of limited pathogenicity (Warren, 1961) and yet are able to confer immunity to reinfection in the rhesus monkey (Smithers, 1962; Hsu, 1969) by maintaining an immunogenic stimulus, persistent females in thioxanthone-treated patients might con- ceivably play a beneficial role when reinfection is likely. Although depreciation of the immune status of patients after effective treatment has only been a subject of speculation, reinfection remains a major threat to the concept of effective mass therapy. In comparison with other anti-schistosomal drugs, thioxanthone treatment may warrant scrutiny in terms of its effect on prevalence and magnitude of reinfection. REFERENCES Azim, M. 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