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6-Trifluoromethyl-2-thiouracil possesses anti-Toxoplasma gondii effect in vitro and in vivo with low hepatotoxicity
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Hwa-Jung Choi a,1, Seung-Taek Yu b,1, Kee-In Lee c, Joong-Kwon Choi c, Bo-Yoon Chang d, Sung-Yeon Kim d, Mi-Hwa Ko e, Hyun-Ok Song a,⇑⇑, Hyun Park a,⇑ a
Department of Infection Biology, Zoonosis Research Center, Wonkwang University School of Medicine, 460 Iksandae-ro, Iksan, Jeonbuk 570-749, Republic of Korea Department of Pediatrics, Wonkwang University School of Medicine, 460 Iksandae-ro, Iksan, Jeonbuk 570-749, Republic of Korea Korea Chemical Bank, Korea Research Institute of Chemical Technology, 141 Gagjeong-ro, Yuseong, Daejeon 305-600, Republic of Korea d Department of Pharmacy, Wonkwang University, 460 Iksandae-ro, Iksan, Jeonbuk 570-749, Republic of Korea e Division of Mathematics and Informational Statistics, Wonkwang University, 460 Iksandae-ro, Iksan, Jeonbuk 570-749, Republic of Korea b c
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h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ATT-5126 and KH-0562 have potent
anti-Toxoplasma gondii activity in vitro. The amount of tachyzoites in mice were decreased by treatment of ATT-5126 and KH-0562. The weights of liver and spleen increased by T. gondii were decreased after treatment of two compounds. The biochemical parameters of liver were restored by KH-0562 treatment. KH-0562 may be a useful candidate for treating T. gondii infection.
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Article history: Received 29 August 2013 Received in revised form 17 March 2014 Accepted 4 May 2014 Available online xxxx Keywords: Toxoplasmosis Anti-Toxoplasma gondii activity KH-0562
a b s t r a c t Since pyrimethamine, the general therapeutic drug for toxoplasmosis, presents several adverse side effects, the need to develop and evaluate new drugs for the condition is critical. In this study, antiToxoplasma gondii activities of 3-[{2-((E)-furan-2-ylmethylene)hydrazinyl}methylene]-1,3-dihydroindol2-one (ATT-5126) and 6-trifluoromethyl-2-thiouracil (KH-0562) were evaluated in vitro using a 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and in vivo by measuring amount of the tachyzoites in mice ascites. Biochemical parameters such as lipid peroxidation (LPO), glutathione (GSH), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were also evaluated in livers of mice at 4 days post-infection. As a result, the ATT-5126 and KH0562 showed anti-T. gondii activity in vitro. Treatment of ATT-5126 or KH-0562 decreased the amount of tachyzoites in T. gondii infected ICR mice. The relative weight of liver and spleen increased by T. gondii infection were decreased by treatment of ATT-5126 or KH-0562. The levels of LPO, ALT and AST, which are biochemical parameters involved in liver injury, were also significantly recovered by treatment of ATT5126 or KH-0562 (p < 0.05). In particular, the recovered levels by KH-0562 were similar to those of pyrimethamine-treated group (p < 0.05). However, the level of GSH, which is an antioxidant indicator, showed insignificant statistics. The results suggest that KH-0562 show anti-T. gondii activities in vitro and in vivo with low hepatotoxicity. Therefore, KH-0562 may be a useful candidate for treating T. gondii infection. Ó 2014 Published by Elsevier Inc.
⇑ Corresponding author. Fax: +82 63 857 0342. ⇑⇑ Co-corresponding author. Fax: +82 63 857 0342. 1
E-mail addresses: [email protected] (H.-O. Song), [email protected] (H. Park). These authors are equally contributed to this work.
http://dx.doi.org/10.1016/j.exppara.2014.05.002 0014-4894/Ó 2014 Published by Elsevier Inc.
Please cite this article in press as: Choi, H.-J., et al. 6-Trifluoromethyl-2-thiouracil possesses anti-Toxoplasma gondii effect in vitro and in vivo with low hepatotoxicity. Exp. Parasitol. (2014), http://dx.doi.org/10.1016/j.exppara.2014.05.002
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1. Introduction The sporozoan parasite Toxoplasma gondii is a tissue parasite common to humans and other animals, particularly those infected with human immune deficiency virus (HIV) (Couzinet et al., 2000; Osunkalu et al., 2011). Humans and other animals are usually infected with T. gondii when they ingest the undercooked meat of infected animals or other food or water contaminated with oocysts (Dubey et al., 2012). The parasite causes toxoplasmosis, which could be life threatening to immune compromised individuals (Luft et al., 1984). The effective drugs used today to treat toxoplasmosis are sulfonamides and pyrimethamines, administered either alone or in synergistic combination, but these (e.g., sulfadiazine) frequently generate adverse side effects such as bone marrow suppression, hypersensitivity, and fever (Jiang et al., 2008). Other drugs (e.g. clindamycin, atovaquone, daspone, trimethoprim, pentamidine, and azithromycin) have been used in the clinic, but relapse is often seen with these because of their common side-effects such as diarrhea, nausea, abdominal pain, cholestatic hepatitis, hepatomegaly and hepatitis (Georgiev, 1994). The liver plays a pivotal role in regulating various physiological processes; it has an enormous capacity to detoxify toxic substances and to synthesize useful principles (Mistry et al., 2013). It plays an important role in glucose and lipid homeostasis (Badole and Bodhankar, 2010). An increase in the levels of liver aspartate aminotransferase (AST) and alanine aminotransferase (ALT) indicate hepatic dysfunction (Ramesh et al., 2012). Therefore, AST and ALT are considered liver toxicity markers (Eliza et al., 2009). Lipid peroxidation is believed to be an important cause of destruction and damage to cell membranes and has been suggested as a factor that contributes to the development of oxygen radical-mediated tissue damage (Koc et al., 2003). The antioxidant system consists of low-molecular-weight antioxidant molecules, such as glutathione (GSH) and various antioxidant enzymes (Sharma and Haldar, 2006). Glutathione is one of the most important molecules playing a role in the cellular defense against chemically reactive toxic compounds that induce oxidative stress, while depletion of tissue GSH is one of the primary factors that permits lipid peroxidation to Q2 occur (Shirazi et al., 2013). Cell-based small-molecule screens could identify new inhibitors for key parasite processes, including invasion, replication, and egress (Carey et al., 2004; Hall et al., 2012; Murakami et al., Q3 2003). We have been searching for anti-Toxoplasma agents from natural and synthetic compounds. A recent article in this field opened the door to the study of simple and efficient model systems for screening anti-Toxoplasma drugs (Jin et al., 2009). Moreover, we reported that oleuropein isolated from Fraxinus rhynchophylla possesses anti-toxoplasmosis effects (Jiang et al., 1994). In this present work, we have applied an in vitro screening procedure to systematically identify synthetic chemicals that exhibit anti-T. gondii activity. Among these, we selected 3-[{2-((E)furan-2-ylmethylene)hydrazinyl}methylene]-1,3-dihydroindol-2-one (ATT-5126) and 6-trifluoromethyl-2-thiouracil (KH-0562) for further evaluation of their anti-T. gondii activity in mice and their effect on the liver biochemical parameters such as LPO, GSH, ALT, and AST.
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2. Materials and methods
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2.1. Cells, reagents and chemicals
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Human epitheloid carcinoma cervix (HeLa) cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained in a Roswell Park Memorial Institute (RPMI)
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1640 medium, supplemented with 10% fetal bovine serum and 0.01% antibiotic–antimycotic. The antibiotic–antimycotic, trypsin–EDTA, fetal bovine serum, and RPMI 1640 medium were supplied by Gibco BRL (Grand Island, NY, USA).The tissue culture plates were purchased from Falcon (BD Biosciences, Franklin Lakes, NJ, USA), and pyrimethamine and dimethyl sulfoxide (DMSO) were purchased from the Sigma–Aldrich Corp. (St. Louis, MO, USA). Pyrimethamine was used as the positive control. The CellTiter 96Ò AQueous One Solution Cell Proliferation Assay kit was obtained from Promega Corporation (Madison, WI, USA) and the3-[{2-((E)furan-2-ylmethylene)hydrazinyl}methylene]-1,3-dihydroindol-2-one (ATT-5126) and 6-trifluoromethyl-2-thiouracil (KH-0562) from Korea Chemical Bank stock. All other chemicals were of reagent grade.
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2.2. Parasite strains and culture
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T. gondii (RH strain) was used for both in vitro and in vivo experiments. T. gondii was cultured in female ICR mice (Koatech, Pyeongtaek-city, South Korea), and tachyzoites were obtained from peritoneal fluids, as previously reported (Jin et al., 2009).
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2.3. Assay of anti-T. gondii activity of ATT-5126 and KH-0562 in vitro
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The anti-T. gondii activity was evaluated by the 3-(4,5-dimethylthiazol-zyl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazoliuzolium, inner salt (MTS) assay using the CellTiter 96Ò AQueous One Solution Cell Proliferation Assay kit, as recently reported by Jin et al. (2009). This assay was described to be very simple for the screen of anti-T. gondii compounds in vitro as it exhibited high correlation with the conventional morphological assay (Giemsa staining). Pyrimethamine was used as the positive control and DMSO as the negative control. HeLa cells were seeded onto a 96-well culture plate at the concentration of 2 104 cells per well. The following day, the medium was removed, the cells were washed with 1 phosphate buffered saline (PBS), and then they were infected with 1 105 T. gondii (host cells:tachyzoites = 1:5) for 24 h. The test chemical or the positive control were treated for 24 h. MTS solution (20 ll) was added directly to the culture wells and these were incubated for 1.5 h at 37 °C. The absorbance at 490 nm was then recorded in a Tecan Infinite F200 microplate reader (Tecan; Männedorf, Switzerland). Cell viability was expressed relatively, as a percentage of the control value. Anti-T. gondii activity was expressed as the selectivity index [SI = concentration required to reduce HeLa cell growth by 50% (CC50)/concentration required to inhibit the T. gondii-induced cytopathic effect by 50% (IC50)].
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2.4. Animals
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Female ICR mice (6 weeks) were purchased from the Koatech company (Pyeongtaek, Korea). The mice were housed and maintained in the animal care facility at Wonkwang University. All experiments with them were performed in accordance with the guidelines approved by the Animal Ethics Committee of our institution.
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2.5. Assay of anti-T. gondii activity of ATT-5126 and KH-0562 in vivo
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ICR female mice (5 mice/group) were infected intraperitoneally with 1 105 tachyzoites. They were separated into 5 groups: the normal group (no T. gondii infection), the negative control group (T. gondii infection and water treatment), the positive control group (T. gondii infection and pyrimethamine treatment), KH-0562 group (T. gondii infection and KH-0562 treatment), and ATT-5126 group (T. gondii infection and ATT-5126 treatment).
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Please cite this article in press as: Choi, H.-J., et al. 6-Trifluoromethyl-2-thiouracil possesses anti-Toxoplasma gondii effect in vitro and in vivo with low hepatotoxicity. Exp. Parasitol. (2014), http://dx.doi.org/10.1016/j.exppara.2014.05.002
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Two hours after infection, the mice in the negative control, positive control, and the 2 drug groups were orally administered with 300 ll of water or 20 mg/kg pyrimethamine or 100 mg/kg KH-0562 or 100 mg/kg ATT-5126, respectively, once daily for 4 days. The animals were sacrificed 4 days post-infection (d.p.i.). Ascites were drawn out to determine the extent of tachyzoite proliferation by counting tachyzoites using a hemocytometer (Murakami et al., 2003).
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2.6. Measurement of LPO, GSH, ALT and AST levels in mouse liver
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The spleens and livers were weighed and then LPO, GSH, ALT, and AST levels were measured. LPO was measured by the thiobarbituric acid reaction, according to the method of Ohkawa et al. (1979). The liver homogenate was added to a mixture of SDS (8.1%, 0.2 ml), thiobarbituric acid (0.67%, 1.5 ml), acetic acid (pH 3.5, 1.5 ml), and water (0.6 ml), and incubated at 95 °C for 1 h, then brought with ice. After an addition of n-butanol (5 ml), the mixture was centrifuged at 4000 rpm for 10 min and the absorbance of the organic layer was measured at 532 nm. Tetraethoxypropane replaced the liver homogenate in the standard sample. The liver glutathione was measured by the enzymatic recycling method (Griffith, 1980). The liver was liquefied in HClO4 (1 M, 8 ml, including 2 mM EDTA) by polytron and the homogenate was centrifuged at 5000 rpm for 5 min. Phosphate buffer (phosphate 0.125 M, EDTA 6.3 mM, pH 7.5), NADPH (0.3 mM, 0.7 ml) and 5,50 -dithio-bis-(2-nitrobenzoic acid) (6 mM, 0.1 ml) were added to the separated supernatant in a semi-microcuvette, mixed, and held at room temperature for 4 min. Glutathione reductase (50 units/ml) was added, and the absorbance measured at 412 nm. Serum levels of AST and ALT were measured according to the method of Reitman and Frankel (1957). The substrate reaction of ALT or AST and serum was carried out under incubation at 37 °C for 30 or 60 min, then 2,4-DNPH was added at room temperature and held for 20 min. Finally, NaOH was added and allowed to react for 30 min. The absorbance at 520 nm was measured.
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Fig. 1. Structures of ATT-5126 and KH-0562. 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221
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2.7. Statistical analysis
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Three independent experiments were performed to analyze in vitro anti-T. gondii activity of chemicals. For the analysis of in vitro anti-T. gondii activity and levels of liver parameters, each measurement was conducted in triplicate. All values are represented as mean ± S.D. Statistical significance was determined using Student’s t-tests for in vitro/in vivo anti-T. gondii activity. The significant differences of LPO, ALT, AST, and GSH levels between drug treated and control mouse groups were analyzed by analysis of variance (ANOVA) and Duncan’s test. p < 0.05 were considered statistically significant.
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3. Results
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3.1. In vitro anti-T. gondii activity of ATT-5126 and KH0562
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In this study, we used a focused screen of compounds that inhibited growth of T. gondii in vitro, and from those compounds, we identified 2 active synthetic compounds, ATT-5126 and KH-0562 (Fig. 1). ATT-5126 showed anti-T. gondii activity with a 50% inhibitory concentration (IC50) of 19.7 lM, a 50% cytotoxicity concentration (CC50) of 35.4 lM, and a derived selectivity index (SI) of 1.8 (Table 1). KH-0562 also manifested anti-T. gondii activity with an IC50 of 32.2 lM, CC50 of 56.3 lM, and SI of 1.7 (Table 1). However, pyrimethamine’s activity was relatively weaker than those of ATT-5126 and KH-0562, with an IC50 of 850.0 lM, CC50 of 760.0 lM, and SI of 0.9 (Table 1).
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Table 1 In vitro anti-Toxoplasma gondii activity of ATT-5126 and KH-0562. Test drug
CC50a
IC50b
SIc
ATT-5126 KH-0562 Pyrimethamine
35.4 ± 0.1 56.3 ± 0.2 760.0 ± 0.1
19.7 ± 0.1 32.2 ± 0.2 850.0 ± 0.4
1.8 1.7 0.9
Results are presented as the mean IC50 and CC50 values obtained from three independent experiments carried out in triplicate ± S.D. ATT-5126, 3-[{2-((E)-furan2-ylmethylene)hydrazinyl}methylene]-1,3-dihydroindol-2-one; KH-0562, 6-trifluoromethyl-2-thiouracil. a Concentration required to reduce HeLa cell growth by 50% (lM). b Concentration required to inhibit T. gondii-induced cytopathic effect by 50% (lM). c Selectivity index = CC50/IC50.
3.2. In vivo anti-T. gondii activity of ATT-5126 and KH-0562
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The mice were sacrificed at 4 d.p.i. and their ascites were used to determine tachyzoite proliferation. The amount of tachyzoites in the peritoneal cavity of the negative group, ATT-0526-treated group, KH-0562-treated group, and pyrimethamine-treated positive group were approximately 1000, 812, 764 and 201 (8.9 104), respectively (Table 2). The amount of tachyzoites in ATT-0526 and KH-0562-treated groups was significantly decreased than that of negative groups (p < 0.05). The inhibition ratios against T. gondii tachyzoite proliferation of the 2 compounds were relatively inferior to that obtained from pyrimethamine treatment.
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3.3. Effect of ATT-5126 and KH-0562 on relative liver and spleen weights
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The relative liver weight of the negative control was just slightly greater than that of the normal group (Fig. 2). Liver weight increased with infection of T. gondii; it was similar to that of the normal group after treatment with ATT-0526, KH-0562, or pyrimethamine (Fig. 2). The relative spleen weight also increased significantly after infection with T. gondii (Fig. 2). Treatment with ATT-0526, KH-0562, or pyrimethamine reduced the increment in spleen weight caused by the T. gondii infection (Fig. 2). However,
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Table 2 In vivo anti-Toxoplasma gondii activity of ATT-5126 and KH-0562. Amount of tachyzoite (8.9 106) N.C.a ATT-5126 KH-0562 Pyrimethamine
1000 ± 21 812 ± 43* 764 ± 12* 201 ± 34*
a T. gondii-infected ICR mice with no treatment; ATT-5126, 3-[{2-((E)-furan-2ylmethylene)hydrazinyl}methylene]-1,3-dihydroindol-2-one; KH-0562, 6-trifluoromethyl-2-thiouracil. * Significantly different from N.C. (p < 0.05).
Please cite this article in press as: Choi, H.-J., et al. 6-Trifluoromethyl-2-thiouracil possesses anti-Toxoplasma gondii effect in vitro and in vivo with low hepatotoxicity. Exp. Parasitol. (2014), http://dx.doi.org/10.1016/j.exppara.2014.05.002
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Fig. 2. Effects of ATT-5126 and KH-0562 on weight change of liver and spleen in T. gondii infected ICR mice. All mice were sacrificed at 4 days post-infection of T. gondii and the relative weights of liver and spleen measured. N.C., Negative control (T. gondii-infected ICR mice with no treatment).
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changes of liver and spleen’s weights didn’t show statistically significant differences.
3.4. Effect of ATT-5126 and KH-0562 on LPO, GSH, ALT and AST levels in mouse liver The effects of ATT-5126 and KH-0562 on liver biochemical parameters are shown in Table 3. LPO served as an indicator of liver damage. The LPO level of the negative group was 121.4 nmol/g, while that of the group treated with 100 mg/kg of KH-0562 and 20 mg/kg of pyrimethamine was significantly decreased to 87.5 nmol/g and 86.5 nmol/g, respectively (p < 0.05, Table 3). The LPO level of the group treated with 100 mg/kg of KH-0562 was comparable to that of pyrimethamine-treated group (87.4 nmol/ g). The LPO level of the group treated with 100 mg/kg of ATT5126 showed relatively weak reduction to 105.2 nmol/g compared to that of the negative group. Glutathione, one of the most important antioxidant molecules in the liver, was measured. In the negative group, it was 4.1 lmol/g (Table 3). Pyrimethamine treatment led to an increase in the glutathione level, to 5.6 lmol/g. In the groups treated with 100 mg/kg of ATT-5126 and 100 mg/kg of KH-0562, the glutathione level reached 3.8 and 3.9 lmol/g, respectively, which are insignificant changes compared to that of negative group (Table 3).
Table 3 Effect of ATT-5126 and KH-0562 on LPO, GSH, ALT and AST levels in T. gondii infected ICR mice. LPO (nmol/g) GSH (lmol/g) ALT (U/ml) Normal 47.5 ± 1.4 N.C.a 121.4 ± 2.4* ATT-5126 105.2 ± 7.8 KH-0562 87.4 ± 9.7** Pyrimethamine 86.5 ± 10.2**
6.5 ± 0.4 4.1 ± 0.7 3.8 ± 0.4 3.9 ± 0.9 5.6 ± 1.4
AST (U/ml)
207.4 ± 4.6 165.4 ± 6.1 2210.4 ± 9.7* 1807.1 ± 4.7* ** 1206.6 ± 6.7 610.4 ± 10.7** 207.6 ± 7.9** 201.1 ± 5.7** 154.3 ± 8.4** 204.3 ± 2.5**
Results are presented as the mean ± S.D. obtained from measurement in triplicate. a T. gondii-infected ICR mice with no treatment; ATT-5126, 3-[{2-((E)-furan-2ylmethylene)hydrazinyl}methylene]-1,3-dihydroindol-2-one; KH-0562, 6-trifluoromethyl-2-thiouracil. * Significantly different from normal (p < 0.05). ** Significantly different from N.C. (p < 0.05).
The levels of ALT and AST as indicators of hepatotoxicity were 2210 and 1807 U/ml, respectively, in the negative group (Table 3). The levels of ALT and AST in the negative group were increased significantly following infection of T. gondii (p < 0.05). The levels of ALT and AST were significantly lower in the pyrimethamine-treated group (154 and 204 U/ml, respectively) compared to those of the negative group (p < 0.05). KH-0562 also significantly recovered the levels of ALT and AST (207 and 201 U/ml, respectively, p < 0.05). The levels were considerably comparable to those of pyrimethamine-treated group. Compared to those of the negative group, treatment with ATT-5126 also significantly decreased the elevated ALT and AST levels to 206 and 610 U/ml, respectively (p < 0.05, Table 3).
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4. Discussion
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T. gondii is an obligate intracellular apicomplexan parasite that infects nucleated cells of birds and mammals. It can cause toxoplasmosis, a disease the leads to ocular and neurological damage, systemic illness, and death (Alves et al., 2012). Available treatments include the antifolates pyrimidine and sulfonamide, but their side effects limit their usefulness (Haverkos, 1987). Novel nontoxic anti-Toxoplasma agents are greatly needed. In this study, we screened ATT-5126 and KH-0562 showing effective anti-T. gondii activity in vitro. Furthermore, in vitro antiT. gondii activity of screened chemicals was considerably stronger than that of pyrimethamine. in vivo treatment with ATT-5126 or KH-0562 significantly decreased the number of tachyzoites in T. gondii-infected ICR mice (p < 0.05). Pyrimethamine treatment in vivo exerted a favorable influence on the level of tachyzoites in T. gondii-infected ICR mice. Although in vivo efficacy of ATT-5126 and KH-0562 was relatively lower than that of pyrimethamine, we ascertained that ATT-5126 and KH-0562 do possess some anti-T. gondii properties. A major target organ for chemical toxicity in laboratory animals is the liver (Boverhof et al., 2005, 2006; Chang et al., 2005). Few studies have completed a systematic analysis of multi-organ involvement in acute, acquired toxoplasmosis (AAT), and the disease can attack the liver, spleen, and retina (Neves et al., 2009). AAT can cause hematological changes (Durlach et al., 2003). Anemia, leucopenia, lymphocytosis, thrombocytopenia, hepatitis, and pneumonia may be secondary to AAT (Neves et al., 2009). In order to address the significance of ATT-5126 or KH-0562 as anti-T. gondii drugs, we investigated the relative weight changes of the liver and spleen in T. gondii-infected ICR mice treated with ATT-5126 or KH-0562. The relative weights of liver and spleen were increased when infected with T. gondii, but they seemed to be decreased after treatment with ATT-5126 or KH-0562. The results demonstrated that the 2 compounds reduced the gain in hepatic weight induced by AAT. Glutathione (GSH) is a key cellular antioxidant that protects cells against damage caused by numerous electrophilic compounds, which include reactive intermediates formed from the metabolism of numerous drugs and a wide range of other xenobiotics present in food and the environment (Ketterer et al., 1983; Meister, 1998; Mitchell et al., 1973). The detoxification of reactive metabolites results in the consumption of glutathione, either via its oxidation to glutathione disulfide or by the formation of conjugation which forms glutathione conjugates (Meister, 1998; Mitchell et al., 1973). In this paper, ATT-5126 or KH-0562 treatment did not show significant changes in GSH level compared to negative group. Therefore, this result indicated that ATT-5126 and KH-0562 are not involved in cellular antioxidant. LPO is believed to be an important cause of destruction and damage to cell membranes; it thought to contribute to the devel-
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opment of oxygen-radical-mediated tissue damage (Koc et al., 2003). AST and ALT were used as biomarkers to evaluate hepatotoxicity (Eliza et al., 2009). We examined AST, ALT and LPO levels. We have shown that the elevation in LPO brought about by T. gondii infection was significantly diminished by treatment with KH-0562 (p < 0.05). We also found that treatment with ATT-5126 or KH-0562 significantly recovered AST and ALT levels compared to the levels in the negative group (p < 0.05). In particular, the recovered levels by KH-0562 were similar to those of pyrimethamine-treated group leading to KH-0562 as a potent anti-T. gondii drug candidate with low hepatotoxicity. Kupffer cells play an important role in the normal physiology and homeostasis of the liver as well as participating in the acute and chronic responses of the liver to toxic compounds and appear to protect against liver injury from the industrial chemicals such as thioacetamide (Andres et al., 2003; Roberts et al., 2006). Intravital microscopy imaging studies have determined that apicomplexan parasites such as T. gondii invade the liver, gradually move toward the surface of sinusoidal epithelial cells to the Kupffer cells, finally entering and developing in the cytoplasm of hepatocytes (Frevert et al., 2005). Indeed, we showed that the negative groups, T. gondii-infected and non-treated group, led to the elevation of LPO, ALT and AST level, confirming that T. gondii infection itself can induce liver injury by invading Kupffer cells. In this article, we revealed that KH-0562 restored the elevated LPO, ALT and AST in negative group with comparable level to pyrimethamine. Thus, it seems that significant recovery of LPO, ALT and AST levels by treatment of KH-0562 is probably caused by the inhibition of hepatotoxicity derived from T. gondii infection. In conclusion, our findings showed the potential of both ATT5126 and KH-0562 to develop novel anti-T. gondii drugs. Especially, KH-0562 restored the levels of LPO, AST, and ALT elevated in the negative control. Although in vivo efficacy of KH-0562 was relatively lower than pyrimethamine, KH-0562 could be a novel drug candidate. Further optimization of compound would be necessary to increase in vivo anti-T. gondii activity.
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Acknowledgments
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Q4 This work was supported by a 2012 grant of Wonkwang UniverQ5 sity. The chemical library used in this study was kindly provided by 391 Korea Chemical Bank of the Korea Research Institute of Chemical 392 Q6 Technology. 389 390
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Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95, 351–358. Osunkalu, V.O., Akanmu, S.A., Nkolika, J.O., Igwebuike, I.V., Adewumi, A.A., Akinde, R.O., Onwuezobe, I.A., 2011. Seroprevalence of Toxoplasma gondii IgG antibody in HIV-infected patients at the Lagos University Teaching Hospital. HIV AIDS (Auckl.) 3, 101–105. Ramesh, B., Karuna, R., Sreenivasa, R.S., Haritha, K., Sai, M.D., Sasi, B.R., Saralakumari, D., 2012. Effect of Commiphora mukul gum resin on hepatic marker enzymes, lipid peroxidation and antioxidants status in pancreas and heart of streptozotocin induced diabetic rats. Asian Pac. J. Trop. Biomed. 2, 895–900. Reitman, S., Frankel, S.K., 1957. A colorimetric method for the determination of serum glutamic oxaloacetic and glutamic pyruvic transaminase. Am. J. Clin. Pathol. 28, 56–63. Roberts, R.A., Ganey, P.E., Ju, C., Kamendulis, L.M., Rusyn, I., Klaunig, J.E., 2006. Role of the Kupffer cell in mediating hepatic toxicity and carcinogenesis. Toxicol. Sci. 96, 2–15. Sharma, S., Haldar, C., 2006. Melatonin prevents X-ray irradiation induced oxidative damage in peripheral blood and spleen of the seasonally breeding rodent, Funambulus pennanti during reproductively active phase. Int. J. Radiat. Biol. 82, 411–419.
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Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr 5 6
6-Trifluoromethyl-2-thiouracil possesses anti-Toxoplasma gondii effect in vitro and in vivo with low hepatotoxicity
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Hwa-Jung Choi a,1, Seung-Taek Yu b,1, Kee-In Lee c, Joong-Kwon Choi c, Bo-Yoon Chang d, Sung-Yeon Kim d, Mi-Hwa Ko e, Hyun-Ok Song a,⇑⇑, Hyun Park a,⇑ a
Department of Infection Biology, Zoonosis Research Center, Wonkwang University School of Medicine, 460 Iksandae-ro, Iksan, Jeonbuk 570-749, Republic of Korea Department of Pediatrics, Wonkwang University School of Medicine, 460 Iksandae-ro, Iksan, Jeonbuk 570-749, Republic of Korea Korea Chemical Bank, Korea Research Institute of Chemical Technology, 141 Gagjeong-ro, Yuseong, Daejeon 305-600, Republic of Korea d Department of Pharmacy, Wonkwang University, 460 Iksandae-ro, Iksan, Jeonbuk 570-749, Republic of Korea e Division of Mathematics and Informational Statistics, Wonkwang University, 460 Iksandae-ro, Iksan, Jeonbuk 570-749, Republic of Korea b c
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h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ATT-5126 and KH-0562 have potent
anti-Toxoplasma gondii activity in vitro. The amount of tachyzoites in mice were decreased by treatment of ATT-5126 and KH-0562. The weights of liver and spleen increased by T. gondii were decreased after treatment of two compounds. The biochemical parameters of liver were restored by KH-0562 treatment. KH-0562 may be a useful candidate for treating T. gondii infection.
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a r t i c l e 3 4 7 8 38 39 40 41 42 43 44 45 46 47
i n f o
Article history: Received 29 August 2013 Received in revised form 17 March 2014 Accepted 4 May 2014 Available online xxxx Keywords: Toxoplasmosis Anti-Toxoplasma gondii activity KH-0562
a b s t r a c t Since pyrimethamine, the general therapeutic drug for toxoplasmosis, presents several adverse side effects, the need to develop and evaluate new drugs for the condition is critical. In this study, antiToxoplasma gondii activities of 3-[{2-((E)-furan-2-ylmethylene)hydrazinyl}methylene]-1,3-dihydroindol2-one (ATT-5126) and 6-trifluoromethyl-2-thiouracil (KH-0562) were evaluated in vitro using a 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and in vivo by measuring amount of the tachyzoites in mice ascites. Biochemical parameters such as lipid peroxidation (LPO), glutathione (GSH), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were also evaluated in livers of mice at 4 days post-infection. As a result, the ATT-5126 and KH0562 showed anti-T. gondii activity in vitro. Treatment of ATT-5126 or KH-0562 decreased the amount of tachyzoites in T. gondii infected ICR mice. The relative weight of liver and spleen increased by T. gondii infection were decreased by treatment of ATT-5126 or KH-0562. The levels of LPO, ALT and AST, which are biochemical parameters involved in liver injury, were also significantly recovered by treatment of ATT5126 or KH-0562 (p < 0.05). In particular, the recovered levels by KH-0562 were similar to those of pyrimethamine-treated group (p < 0.05). However, the level of GSH, which is an antioxidant indicator, showed insignificant statistics. The results suggest that KH-0562 show anti-T. gondii activities in vitro and in vivo with low hepatotoxicity. Therefore, KH-0562 may be a useful candidate for treating T. gondii infection. Ó 2014 Published by Elsevier Inc.
⇑ Corresponding author. Fax: +82 63 857 0342. ⇑⇑ Co-corresponding author. Fax: +82 63 857 0342. 1
E-mail addresses: [email protected] (H.-O. Song), [email protected] (H. Park). These authors are equally contributed to this work.
http://dx.doi.org/10.1016/j.exppara.2014.05.002 0014-4894/Ó 2014 Published by Elsevier Inc.
Please cite this article in press as: Choi, H.-J., et al. 6-Trifluoromethyl-2-thiouracil possesses anti-Toxoplasma gondii effect in vitro and in vivo with low hepatotoxicity. Exp. Parasitol. (2014), http://dx.doi.org/10.1016/j.exppara.2014.05.002
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1. Introduction The sporozoan parasite Toxoplasma gondii is a tissue parasite common to humans and other animals, particularly those infected with human immune deficiency virus (HIV) (Couzinet et al., 2000; Osunkalu et al., 2011). Humans and other animals are usually infected with T. gondii when they ingest the undercooked meat of infected animals or other food or water contaminated with oocysts (Dubey et al., 2012). The parasite causes toxoplasmosis, which could be life threatening to immune compromised individuals (Luft et al., 1984). The effective drugs used today to treat toxoplasmosis are sulfonamides and pyrimethamines, administered either alone or in synergistic combination, but these (e.g., sulfadiazine) frequently generate adverse side effects such as bone marrow suppression, hypersensitivity, and fever (Jiang et al., 2008). Other drugs (e.g. clindamycin, atovaquone, daspone, trimethoprim, pentamidine, and azithromycin) have been used in the clinic, but relapse is often seen with these because of their common side-effects such as diarrhea, nausea, abdominal pain, cholestatic hepatitis, hepatomegaly and hepatitis (Georgiev, 1994). The liver plays a pivotal role in regulating various physiological processes; it has an enormous capacity to detoxify toxic substances and to synthesize useful principles (Mistry et al., 2013). It plays an important role in glucose and lipid homeostasis (Badole and Bodhankar, 2010). An increase in the levels of liver aspartate aminotransferase (AST) and alanine aminotransferase (ALT) indicate hepatic dysfunction (Ramesh et al., 2012). Therefore, AST and ALT are considered liver toxicity markers (Eliza et al., 2009). Lipid peroxidation is believed to be an important cause of destruction and damage to cell membranes and has been suggested as a factor that contributes to the development of oxygen radical-mediated tissue damage (Koc et al., 2003). The antioxidant system consists of low-molecular-weight antioxidant molecules, such as glutathione (GSH) and various antioxidant enzymes (Sharma and Haldar, 2006). Glutathione is one of the most important molecules playing a role in the cellular defense against chemically reactive toxic compounds that induce oxidative stress, while depletion of tissue GSH is one of the primary factors that permits lipid peroxidation to Q2 occur (Shirazi et al., 2013). Cell-based small-molecule screens could identify new inhibitors for key parasite processes, including invasion, replication, and egress (Carey et al., 2004; Hall et al., 2012; Murakami et al., Q3 2003). We have been searching for anti-Toxoplasma agents from natural and synthetic compounds. A recent article in this field opened the door to the study of simple and efficient model systems for screening anti-Toxoplasma drugs (Jin et al., 2009). Moreover, we reported that oleuropein isolated from Fraxinus rhynchophylla possesses anti-toxoplasmosis effects (Jiang et al., 1994). In this present work, we have applied an in vitro screening procedure to systematically identify synthetic chemicals that exhibit anti-T. gondii activity. Among these, we selected 3-[{2-((E)furan-2-ylmethylene)hydrazinyl}methylene]-1,3-dihydroindol-2-one (ATT-5126) and 6-trifluoromethyl-2-thiouracil (KH-0562) for further evaluation of their anti-T. gondii activity in mice and their effect on the liver biochemical parameters such as LPO, GSH, ALT, and AST.
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2. Materials and methods
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2.1. Cells, reagents and chemicals
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Human epitheloid carcinoma cervix (HeLa) cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained in a Roswell Park Memorial Institute (RPMI)
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1640 medium, supplemented with 10% fetal bovine serum and 0.01% antibiotic–antimycotic. The antibiotic–antimycotic, trypsin–EDTA, fetal bovine serum, and RPMI 1640 medium were supplied by Gibco BRL (Grand Island, NY, USA).The tissue culture plates were purchased from Falcon (BD Biosciences, Franklin Lakes, NJ, USA), and pyrimethamine and dimethyl sulfoxide (DMSO) were purchased from the Sigma–Aldrich Corp. (St. Louis, MO, USA). Pyrimethamine was used as the positive control. The CellTiter 96Ò AQueous One Solution Cell Proliferation Assay kit was obtained from Promega Corporation (Madison, WI, USA) and the3-[{2-((E)furan-2-ylmethylene)hydrazinyl}methylene]-1,3-dihydroindol-2-one (ATT-5126) and 6-trifluoromethyl-2-thiouracil (KH-0562) from Korea Chemical Bank stock. All other chemicals were of reagent grade.
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2.2. Parasite strains and culture
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T. gondii (RH strain) was used for both in vitro and in vivo experiments. T. gondii was cultured in female ICR mice (Koatech, Pyeongtaek-city, South Korea), and tachyzoites were obtained from peritoneal fluids, as previously reported (Jin et al., 2009).
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2.3. Assay of anti-T. gondii activity of ATT-5126 and KH-0562 in vitro
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The anti-T. gondii activity was evaluated by the 3-(4,5-dimethylthiazol-zyl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazoliuzolium, inner salt (MTS) assay using the CellTiter 96Ò AQueous One Solution Cell Proliferation Assay kit, as recently reported by Jin et al. (2009). This assay was described to be very simple for the screen of anti-T. gondii compounds in vitro as it exhibited high correlation with the conventional morphological assay (Giemsa staining). Pyrimethamine was used as the positive control and DMSO as the negative control. HeLa cells were seeded onto a 96-well culture plate at the concentration of 2 104 cells per well. The following day, the medium was removed, the cells were washed with 1 phosphate buffered saline (PBS), and then they were infected with 1 105 T. gondii (host cells:tachyzoites = 1:5) for 24 h. The test chemical or the positive control were treated for 24 h. MTS solution (20 ll) was added directly to the culture wells and these were incubated for 1.5 h at 37 °C. The absorbance at 490 nm was then recorded in a Tecan Infinite F200 microplate reader (Tecan; Männedorf, Switzerland). Cell viability was expressed relatively, as a percentage of the control value. Anti-T. gondii activity was expressed as the selectivity index [SI = concentration required to reduce HeLa cell growth by 50% (CC50)/concentration required to inhibit the T. gondii-induced cytopathic effect by 50% (IC50)].
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2.4. Animals
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Female ICR mice (6 weeks) were purchased from the Koatech company (Pyeongtaek, Korea). The mice were housed and maintained in the animal care facility at Wonkwang University. All experiments with them were performed in accordance with the guidelines approved by the Animal Ethics Committee of our institution.
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2.5. Assay of anti-T. gondii activity of ATT-5126 and KH-0562 in vivo
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ICR female mice (5 mice/group) were infected intraperitoneally with 1 105 tachyzoites. They were separated into 5 groups: the normal group (no T. gondii infection), the negative control group (T. gondii infection and water treatment), the positive control group (T. gondii infection and pyrimethamine treatment), KH-0562 group (T. gondii infection and KH-0562 treatment), and ATT-5126 group (T. gondii infection and ATT-5126 treatment).
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Two hours after infection, the mice in the negative control, positive control, and the 2 drug groups were orally administered with 300 ll of water or 20 mg/kg pyrimethamine or 100 mg/kg KH-0562 or 100 mg/kg ATT-5126, respectively, once daily for 4 days. The animals were sacrificed 4 days post-infection (d.p.i.). Ascites were drawn out to determine the extent of tachyzoite proliferation by counting tachyzoites using a hemocytometer (Murakami et al., 2003).
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2.6. Measurement of LPO, GSH, ALT and AST levels in mouse liver
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The spleens and livers were weighed and then LPO, GSH, ALT, and AST levels were measured. LPO was measured by the thiobarbituric acid reaction, according to the method of Ohkawa et al. (1979). The liver homogenate was added to a mixture of SDS (8.1%, 0.2 ml), thiobarbituric acid (0.67%, 1.5 ml), acetic acid (pH 3.5, 1.5 ml), and water (0.6 ml), and incubated at 95 °C for 1 h, then brought with ice. After an addition of n-butanol (5 ml), the mixture was centrifuged at 4000 rpm for 10 min and the absorbance of the organic layer was measured at 532 nm. Tetraethoxypropane replaced the liver homogenate in the standard sample. The liver glutathione was measured by the enzymatic recycling method (Griffith, 1980). The liver was liquefied in HClO4 (1 M, 8 ml, including 2 mM EDTA) by polytron and the homogenate was centrifuged at 5000 rpm for 5 min. Phosphate buffer (phosphate 0.125 M, EDTA 6.3 mM, pH 7.5), NADPH (0.3 mM, 0.7 ml) and 5,50 -dithio-bis-(2-nitrobenzoic acid) (6 mM, 0.1 ml) were added to the separated supernatant in a semi-microcuvette, mixed, and held at room temperature for 4 min. Glutathione reductase (50 units/ml) was added, and the absorbance measured at 412 nm. Serum levels of AST and ALT were measured according to the method of Reitman and Frankel (1957). The substrate reaction of ALT or AST and serum was carried out under incubation at 37 °C for 30 or 60 min, then 2,4-DNPH was added at room temperature and held for 20 min. Finally, NaOH was added and allowed to react for 30 min. The absorbance at 520 nm was measured.
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Fig. 1. Structures of ATT-5126 and KH-0562. 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221
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2.7. Statistical analysis
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Three independent experiments were performed to analyze in vitro anti-T. gondii activity of chemicals. For the analysis of in vitro anti-T. gondii activity and levels of liver parameters, each measurement was conducted in triplicate. All values are represented as mean ± S.D. Statistical significance was determined using Student’s t-tests for in vitro/in vivo anti-T. gondii activity. The significant differences of LPO, ALT, AST, and GSH levels between drug treated and control mouse groups were analyzed by analysis of variance (ANOVA) and Duncan’s test. p < 0.05 were considered statistically significant.
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3. Results
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3.1. In vitro anti-T. gondii activity of ATT-5126 and KH0562
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In this study, we used a focused screen of compounds that inhibited growth of T. gondii in vitro, and from those compounds, we identified 2 active synthetic compounds, ATT-5126 and KH-0562 (Fig. 1). ATT-5126 showed anti-T. gondii activity with a 50% inhibitory concentration (IC50) of 19.7 lM, a 50% cytotoxicity concentration (CC50) of 35.4 lM, and a derived selectivity index (SI) of 1.8 (Table 1). KH-0562 also manifested anti-T. gondii activity with an IC50 of 32.2 lM, CC50 of 56.3 lM, and SI of 1.7 (Table 1). However, pyrimethamine’s activity was relatively weaker than those of ATT-5126 and KH-0562, with an IC50 of 850.0 lM, CC50 of 760.0 lM, and SI of 0.9 (Table 1).
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Table 1 In vitro anti-Toxoplasma gondii activity of ATT-5126 and KH-0562. Test drug
CC50a
IC50b
SIc
ATT-5126 KH-0562 Pyrimethamine
35.4 ± 0.1 56.3 ± 0.2 760.0 ± 0.1
19.7 ± 0.1 32.2 ± 0.2 850.0 ± 0.4
1.8 1.7 0.9
Results are presented as the mean IC50 and CC50 values obtained from three independent experiments carried out in triplicate ± S.D. ATT-5126, 3-[{2-((E)-furan2-ylmethylene)hydrazinyl}methylene]-1,3-dihydroindol-2-one; KH-0562, 6-trifluoromethyl-2-thiouracil. a Concentration required to reduce HeLa cell growth by 50% (lM). b Concentration required to inhibit T. gondii-induced cytopathic effect by 50% (lM). c Selectivity index = CC50/IC50.
3.2. In vivo anti-T. gondii activity of ATT-5126 and KH-0562
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The mice were sacrificed at 4 d.p.i. and their ascites were used to determine tachyzoite proliferation. The amount of tachyzoites in the peritoneal cavity of the negative group, ATT-0526-treated group, KH-0562-treated group, and pyrimethamine-treated positive group were approximately 1000, 812, 764 and 201 (8.9 104), respectively (Table 2). The amount of tachyzoites in ATT-0526 and KH-0562-treated groups was significantly decreased than that of negative groups (p < 0.05). The inhibition ratios against T. gondii tachyzoite proliferation of the 2 compounds were relatively inferior to that obtained from pyrimethamine treatment.
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3.3. Effect of ATT-5126 and KH-0562 on relative liver and spleen weights
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The relative liver weight of the negative control was just slightly greater than that of the normal group (Fig. 2). Liver weight increased with infection of T. gondii; it was similar to that of the normal group after treatment with ATT-0526, KH-0562, or pyrimethamine (Fig. 2). The relative spleen weight also increased significantly after infection with T. gondii (Fig. 2). Treatment with ATT-0526, KH-0562, or pyrimethamine reduced the increment in spleen weight caused by the T. gondii infection (Fig. 2). However,
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Table 2 In vivo anti-Toxoplasma gondii activity of ATT-5126 and KH-0562. Amount of tachyzoite (8.9 106) N.C.a ATT-5126 KH-0562 Pyrimethamine
1000 ± 21 812 ± 43* 764 ± 12* 201 ± 34*
a T. gondii-infected ICR mice with no treatment; ATT-5126, 3-[{2-((E)-furan-2ylmethylene)hydrazinyl}methylene]-1,3-dihydroindol-2-one; KH-0562, 6-trifluoromethyl-2-thiouracil. * Significantly different from N.C. (p < 0.05).
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Fig. 2. Effects of ATT-5126 and KH-0562 on weight change of liver and spleen in T. gondii infected ICR mice. All mice were sacrificed at 4 days post-infection of T. gondii and the relative weights of liver and spleen measured. N.C., Negative control (T. gondii-infected ICR mice with no treatment).
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changes of liver and spleen’s weights didn’t show statistically significant differences.
3.4. Effect of ATT-5126 and KH-0562 on LPO, GSH, ALT and AST levels in mouse liver The effects of ATT-5126 and KH-0562 on liver biochemical parameters are shown in Table 3. LPO served as an indicator of liver damage. The LPO level of the negative group was 121.4 nmol/g, while that of the group treated with 100 mg/kg of KH-0562 and 20 mg/kg of pyrimethamine was significantly decreased to 87.5 nmol/g and 86.5 nmol/g, respectively (p < 0.05, Table 3). The LPO level of the group treated with 100 mg/kg of KH-0562 was comparable to that of pyrimethamine-treated group (87.4 nmol/ g). The LPO level of the group treated with 100 mg/kg of ATT5126 showed relatively weak reduction to 105.2 nmol/g compared to that of the negative group. Glutathione, one of the most important antioxidant molecules in the liver, was measured. In the negative group, it was 4.1 lmol/g (Table 3). Pyrimethamine treatment led to an increase in the glutathione level, to 5.6 lmol/g. In the groups treated with 100 mg/kg of ATT-5126 and 100 mg/kg of KH-0562, the glutathione level reached 3.8 and 3.9 lmol/g, respectively, which are insignificant changes compared to that of negative group (Table 3).
Table 3 Effect of ATT-5126 and KH-0562 on LPO, GSH, ALT and AST levels in T. gondii infected ICR mice. LPO (nmol/g) GSH (lmol/g) ALT (U/ml) Normal 47.5 ± 1.4 N.C.a 121.4 ± 2.4* ATT-5126 105.2 ± 7.8 KH-0562 87.4 ± 9.7** Pyrimethamine 86.5 ± 10.2**
6.5 ± 0.4 4.1 ± 0.7 3.8 ± 0.4 3.9 ± 0.9 5.6 ± 1.4
AST (U/ml)
207.4 ± 4.6 165.4 ± 6.1 2210.4 ± 9.7* 1807.1 ± 4.7* ** 1206.6 ± 6.7 610.4 ± 10.7** 207.6 ± 7.9** 201.1 ± 5.7** 154.3 ± 8.4** 204.3 ± 2.5**
Results are presented as the mean ± S.D. obtained from measurement in triplicate. a T. gondii-infected ICR mice with no treatment; ATT-5126, 3-[{2-((E)-furan-2ylmethylene)hydrazinyl}methylene]-1,3-dihydroindol-2-one; KH-0562, 6-trifluoromethyl-2-thiouracil. * Significantly different from normal (p < 0.05). ** Significantly different from N.C. (p < 0.05).
The levels of ALT and AST as indicators of hepatotoxicity were 2210 and 1807 U/ml, respectively, in the negative group (Table 3). The levels of ALT and AST in the negative group were increased significantly following infection of T. gondii (p < 0.05). The levels of ALT and AST were significantly lower in the pyrimethamine-treated group (154 and 204 U/ml, respectively) compared to those of the negative group (p < 0.05). KH-0562 also significantly recovered the levels of ALT and AST (207 and 201 U/ml, respectively, p < 0.05). The levels were considerably comparable to those of pyrimethamine-treated group. Compared to those of the negative group, treatment with ATT-5126 also significantly decreased the elevated ALT and AST levels to 206 and 610 U/ml, respectively (p < 0.05, Table 3).
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4. Discussion
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T. gondii is an obligate intracellular apicomplexan parasite that infects nucleated cells of birds and mammals. It can cause toxoplasmosis, a disease the leads to ocular and neurological damage, systemic illness, and death (Alves et al., 2012). Available treatments include the antifolates pyrimidine and sulfonamide, but their side effects limit their usefulness (Haverkos, 1987). Novel nontoxic anti-Toxoplasma agents are greatly needed. In this study, we screened ATT-5126 and KH-0562 showing effective anti-T. gondii activity in vitro. Furthermore, in vitro antiT. gondii activity of screened chemicals was considerably stronger than that of pyrimethamine. in vivo treatment with ATT-5126 or KH-0562 significantly decreased the number of tachyzoites in T. gondii-infected ICR mice (p < 0.05). Pyrimethamine treatment in vivo exerted a favorable influence on the level of tachyzoites in T. gondii-infected ICR mice. Although in vivo efficacy of ATT-5126 and KH-0562 was relatively lower than that of pyrimethamine, we ascertained that ATT-5126 and KH-0562 do possess some anti-T. gondii properties. A major target organ for chemical toxicity in laboratory animals is the liver (Boverhof et al., 2005, 2006; Chang et al., 2005). Few studies have completed a systematic analysis of multi-organ involvement in acute, acquired toxoplasmosis (AAT), and the disease can attack the liver, spleen, and retina (Neves et al., 2009). AAT can cause hematological changes (Durlach et al., 2003). Anemia, leucopenia, lymphocytosis, thrombocytopenia, hepatitis, and pneumonia may be secondary to AAT (Neves et al., 2009). In order to address the significance of ATT-5126 or KH-0562 as anti-T. gondii drugs, we investigated the relative weight changes of the liver and spleen in T. gondii-infected ICR mice treated with ATT-5126 or KH-0562. The relative weights of liver and spleen were increased when infected with T. gondii, but they seemed to be decreased after treatment with ATT-5126 or KH-0562. The results demonstrated that the 2 compounds reduced the gain in hepatic weight induced by AAT. Glutathione (GSH) is a key cellular antioxidant that protects cells against damage caused by numerous electrophilic compounds, which include reactive intermediates formed from the metabolism of numerous drugs and a wide range of other xenobiotics present in food and the environment (Ketterer et al., 1983; Meister, 1998; Mitchell et al., 1973). The detoxification of reactive metabolites results in the consumption of glutathione, either via its oxidation to glutathione disulfide or by the formation of conjugation which forms glutathione conjugates (Meister, 1998; Mitchell et al., 1973). In this paper, ATT-5126 or KH-0562 treatment did not show significant changes in GSH level compared to negative group. Therefore, this result indicated that ATT-5126 and KH-0562 are not involved in cellular antioxidant. LPO is believed to be an important cause of destruction and damage to cell membranes; it thought to contribute to the devel-
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Please cite this article in press as: Choi, H.-J., et al. 6-Trifluoromethyl-2-thiouracil possesses anti-Toxoplasma gondii effect in vitro and in vivo with low hepatotoxicity. Exp. Parasitol. (2014), http://dx.doi.org/10.1016/j.exppara.2014.05.002
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opment of oxygen-radical-mediated tissue damage (Koc et al., 2003). AST and ALT were used as biomarkers to evaluate hepatotoxicity (Eliza et al., 2009). We examined AST, ALT and LPO levels. We have shown that the elevation in LPO brought about by T. gondii infection was significantly diminished by treatment with KH-0562 (p < 0.05). We also found that treatment with ATT-5126 or KH-0562 significantly recovered AST and ALT levels compared to the levels in the negative group (p < 0.05). In particular, the recovered levels by KH-0562 were similar to those of pyrimethamine-treated group leading to KH-0562 as a potent anti-T. gondii drug candidate with low hepatotoxicity. Kupffer cells play an important role in the normal physiology and homeostasis of the liver as well as participating in the acute and chronic responses of the liver to toxic compounds and appear to protect against liver injury from the industrial chemicals such as thioacetamide (Andres et al., 2003; Roberts et al., 2006). Intravital microscopy imaging studies have determined that apicomplexan parasites such as T. gondii invade the liver, gradually move toward the surface of sinusoidal epithelial cells to the Kupffer cells, finally entering and developing in the cytoplasm of hepatocytes (Frevert et al., 2005). Indeed, we showed that the negative groups, T. gondii-infected and non-treated group, led to the elevation of LPO, ALT and AST level, confirming that T. gondii infection itself can induce liver injury by invading Kupffer cells. In this article, we revealed that KH-0562 restored the elevated LPO, ALT and AST in negative group with comparable level to pyrimethamine. Thus, it seems that significant recovery of LPO, ALT and AST levels by treatment of KH-0562 is probably caused by the inhibition of hepatotoxicity derived from T. gondii infection. In conclusion, our findings showed the potential of both ATT5126 and KH-0562 to develop novel anti-T. gondii drugs. Especially, KH-0562 restored the levels of LPO, AST, and ALT elevated in the negative control. Although in vivo efficacy of KH-0562 was relatively lower than pyrimethamine, KH-0562 could be a novel drug candidate. Further optimization of compound would be necessary to increase in vivo anti-T. gondii activity.
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Acknowledgments
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Q4 This work was supported by a 2012 grant of Wonkwang UniverQ5 sity. The chemical library used in this study was kindly provided by 391 Korea Chemical Bank of the Korea Research Institute of Chemical 392 Q6 Technology. 389 390
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