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Comparative cytotoxicity of N-substituted N′-(4-imidazole-ethyl)thiourea in precision-cut rat liver slices
Toxicology 197 (2004) 81–91
Comparative cytotoxicity of N-substituted N-(4-imidazole-ethyl)thiourea in precision-cut rat liver slices Rob C.A. Onderwater, Jan N.M. Commandeur, Nico P.E. Vermeulen∗ Leiden/Amsterdam Center for Drug Research (LACDR), Division of Molecular Toxicology, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands Received in revised form 17 November 2003; accepted 17 November 2003
Abstract In order to more rationally design thiourea-containing drugs and drug candidates, specifically thiourea-containing histamine H3 receptor antagonists, it is necessary to develop structure–toxicity relationships (STRs). For this purpose, the cytotoxicity of a series of thiourea-containing compounds was tested in precision-cut rat liver slices. A concentration of 1000 M of N-p-bromophenyl, N -(4-imidazole-ethyl)thiourea (8) or N-p-nitrophenyl, N -(4-imidazole-ethyl)thiourea (9) was found to cause cytotoxicity, evidenced as LDH leakage, resulting in more than 95% LDH leakage after 6 h. N-p-Methoxyphenyl, N -(4-imidazole-ethyl)thiourea (6) caused 40.6 ± 19.7% LDH leakage after 6 h. Control levels of cell death (1% methanol as control vehicle) were below 20% in 6 h. After 6 h of exposure, N-p-chlorophenyl, N -(4-imidazole-ethyl)thiourea (7), 8, and 9 were already found to cause significant cytotoxicity at a concentration of 100 M. At 200 M, 9 was found to cause significantly more cytotoxicity than 7 and 8. N-Naphthyl, N -(4-imidazole-ethyl)thiourea (12) was found to cause significant cytotoxicity towards precision-cut rat liver slices after 6 h of exposure to a concentration of 500 M. All other N-substituted, N -(4-imidazole-ethyl)thiourea tested in this study were not found to be cytotoxic towards precision-cut rat liver slices within the 6 h of exposure up to a concentration of 1000 M. Intracellular glutathione (GSH) content and mitochondrial MTT reduction activity were also examined after exposure of slices to N-substituted, N -(4-imidazole-ethyl)thiourea. Both of these markers, however, were not found to provide additional information regarding the possible mechanisms of cytotoxicity, i.e. GSH depletion or reduced mitochondrial activity since these markers did not clearly precede LDH leakage. A correlation was found between cytotoxicity towards precision-cut rat liver slices and Vmax /Km values for the formation of sulfenic acids from N-substituted N -(4-imidazole-ethyl)thiourea by hepatic rat flavin-containing monooxygenases (FMO). The compound with the highest Vmax /Km value for the formation of sulfenic acids, 9, was also the most cytotoxic. Compounds with a significantly lower Vmax /Km value, 7, 8, and 12, were less cytotoxic than 9. Compounds with a Vmax /Km value for the formation of sulfenic acids lower than 0.0788 ml/(min mg) were found not to be cytotoxic towards precision-cut rat liver slices for concentrations up to 1000 M at an exposure time of 6 h. It is concluded, from this study, that N-phenyl substituted N -(4-imidazole-ethyl)thiourea-containing electron-withdrawing p-substituents are cytotoxic towards precision-cut rat liver slices. Cytotoxicity is increased with increasing electron-withdrawing
∗ Corresponding author. Tel.: +31-20-4447590; fax: +31-20-4447610. E-mail address: [email protected] (N.P.E. Vermeulen).
0300-483X/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2003.11.014
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capacity of the p-substituent. A correlation was found to exist between Vmax /Km value for the formation of sulfenic acids by rat liver FMO enzymes and cytotoxicity. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Comparative cytotoxicity; N -(4-Imidazole-ethyl)thiourea; Precision-cut rat liver slices
1. Introduction Interest in the thiourea group as a pharmacophore has rekindled with the development of some promising new thiourea-containing drugs candidates. Among these are potent HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTI). Trovirdine was the first thiourea-containing compound of this class to reach Phase I clinical trails. It was developed through rational design from a series of N-phenylethyl, N thiazolylthiourea (PETT) by Eli Lilly and Medivir (Cantrell et al., 1996; Pedersen and Pedersen, 1999; Högberg and Morrison, 2000). Using a computer model of HIV-1 reverse transcriptase thioureacontaining compounds were designed that show even better activity against the wild type reverse transcriptase as trovirdine and are specifically designed to also strongly inhibit the most often observed resistant strains (Mao et al., 1999; Uckun et al., 2000; Venkatachalam et al., 2000). Several of the newly designed PETT-class NNRTIs have also been shown to have spermicidal activity in the low micromolar range creating. This combination gives these compounds a unique clinical potential of becoming the active ingredients of a vaginal contraceptive for women who are at high risk for acquiring an HIV infection (D’Cruz et al., 2002, 2003). Another interesting lead for drug development can be found among centrally active histamine H3 antagonists (Vollinga et al., 1995). Many of the latter compounds are analogues of one of the first thiourea-containing drugs considered for widespread use, namely the histamine H2 receptor antagonist burimamide. However, its application was never a success, due to its low bioavailability as well as the severe side effects which accompanied its use (Forrest et al., 1975). Thiourea-containing compounds are often goitrogenic and inhibitory towards thyroid hormone biosynthesis, and some cause hypersensitivity reac-
tions and are pulmonary or hepatotoxins (Skellern, 1989). Most adverse reactions are attributed to the thiocarbonyl moiety since in many cases the corresponding urea compounds do not cause similar toxicity (Skellern, 1989; Onderwater et al., 1998). The majority of the studies into thiourea toxicity have been performed with the pulmonary toxin ␣naphthylthiourea. Early radioactivity studies in the 1970s with ␣-naphthylthiourea have shown that both the thionocarbon- and the thionosulfur-atom are covalently bound to rat lung microsomal proteins upon bioactivation (Boyd and Neal, 1976). Oxidation of the thionosulfur moiety is generally accepted to be the first step in thiourea bioactivation. It is generally believed that flavin-containing monooxygenases (FMOs) are responsible for this oxidation, which results in the formation of thiolreactive sulfenic acids (Poulsen et al., 1979; Guo and Ziegler, 1991; Guo et al., 1992; Smith and Crespi, 2002). In order to achieve a rational design of thioureacontaining drugs and drug candidates, more specifically thiourea-containing histamine H3 receptor antagonists, it is necessary to develop structure– toxicity relationships (STRs) of thiourea-containing compounds. For this purpose, the cytotoxicity of a series of 13 thiourea-containing compounds was tested in precision-cut rat liver slices. In order to develop STRs which can directly be applied to histamine antagonists, like burimamide, compounds were synthesized which closely resemble burimamide, without being pharmacologically active. Thus, the pharmacological activity of histamine antagonists was prevented from interfering with the occurrence of cytotoxity in precision-cut rat liver slices. Therefore, N-substituted N -(4-imidazole-ethyl)thiourea, short-chain analogues of burimamide (N-methyl, N (4-imidazole-butyl)thiourea) were used to develop STRs.
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2. Materials and methods 2.1. Chemicals 5,5 -Dithiobis(2-nitrobenzoic acid) (DTNB) was obtained from Fluka Chemie AG (Buchs, Switzerland). NADPH was obtained from Boehringer (Mannheim, Germany), N-ethylmaleimide (NEM) from Aldrich (Steinheim, Germany) and acetylthiocholine from Sigma (St. Louis, USA). N-substituted N -(4-imidazole-ethyl)thiourea were synthesized according to Van der Goot et al. (1992). All other chemicals were of the highest grade commercially available. 2.2. Preparation of precision-cut rat liver slices Rat liver slices were prepared using a Krumdieck Tissue Slicer (Alabama Research and Development). Livers were isolated from male Wistar rats (200– 250 g), which were obtained from Harlan (Zeist, The Netherlands) and which were fed a standard laboratory diet from Hope Farms (Woerden, The Netherlands) and had access to food and water ad libitum. Rats were sacrificed by decapitation and livers were quickly removed. Excised livers were stored at 4 ◦ C in Krebs–Henseleit buffer (pH 7.40) containing 2 g dsglucose/l, which was saturated with carbogen (95% O2 /5% CO2 ), (KH buffer) until cylindrical cores were made with a 5 mm coring tool (Alabama Research and Development). Slices of 250–300 m were made at 4 ◦ C in KH buffer within 30 min of liver isolation. 2.3. Incubation of precision-cut rat liver slices Slices were transferred, in sets of five, to 20 ml polypropylene scintillation vials, containing 3 ml ice-cold KH buffer. Vials were saturated with carbogen and, subsequently, were placed in a rotating incubation block set at 37 ◦ C. Slices were, thus, pre-incubated for 60 mm at 100 rotations per minute. After pre-incubation, single slices were transferred to 20 ml polypropylene scintillation vials, containing 1.5 ml KH buffer of 37 ◦ C. Vials were saturated with carbogen and were subsequently placed in an incubation block set at 37 ◦ C. Test compounds, dissolved in 50% 0.1 M potassium phosphate buffer (pH 7.40)/50% methanol, were added to the vials prior to addition of the slice. After addition of the compounds all incubations contained 1% methanol.
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The carbogen atmosphere was refreshed every 30 min throughout the experiments. At specific timepoints, rat liver slices were transferred from the incubation to Eppendorf vials containing 1 ml ice-cold phosphate buffered saline (PBS) and samples of 1 ml were taken from the incubation medium. Slices were homogenized at 4 ◦ C in a Potter–Ehlvejem tube. A sample of the slice homogenate was taken for protein determination, which was determined with the BIORAD protein reagent, using BSA as a standard, on a Packard Argus 400 microplate reader. 2.4. LDH leakage from precision-cut rat liver slices LDH leakage was determined in both incubation media and slice homogenates by measuring oxidation of NADH in the presence of sodium pyruvate at 340 nm using a Phillips PU-8720 UV-Vis spectrophotometer (Van der Straat et al., 1987). The final reaction mixture (volume 1 ml) contained 0.2 mM NADH and 0.5 mM sodium pyruvate in 50 mM triethanolamine buffer, pH 7.4. Addition of 100 l of the samples initiated the enzymic reaction. LDH leakage from the rat liver slices was calculated from the activities found in total incubation media and slice homogenates. 2.5. Intracellular GSH levels in precision-cut rat liver slices Total glutathione concentrations (GSH and GSSG) in both incubation media and slice homogenates were determined, using a Packard Argus 400 microplate reader, via an enzymatic recycling reaction of GSH in combination with a chromogenic reaction of DTNB, continuously leading to the formation of 5-thio-2-nitrobenzoate which were measured at 405 nm (Redegeld et al., 1988). Oxidized glutathione (GSSG) was determined analogously to the total glutathione measurement, except that GSH was first depleted through reaction with an excess N-ethylmale¨ımide. GSH levels were deduced from total glutathione minus GSSG levels. 2.6. MTT reduction by precision-cut rat liver slices MTT reduction activity was determined according to Lomonte et al. (1993). Slice homogenates were centrifuged for 10 min at 1000 × g. To 250 l of the
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supernatant 750 l of PBS and 200 l of 2.5 mg/ml MTT in PBS were added. The mixture was incubated in polypropylene vials in the dark for 90 min at 37 ◦ C. The yellow colored MTT is reduced to a purple formazan. The difference in absorbance at 570 and 690 nm was determined. 2.7. Thiourea-dependent oxidation of thiocholine by rat liver FMO Thiocholine was prepared from acetylthiocholine in acidic methanol according to Guo and Ziegler (1991). Thiourea-dependent oxidation of thiocholine was determined according to Guo and Ziegler (1991). Rat liver microsomes were prepared as described by Van der Straat et al. (1987). Microsomal protein (0.5 mg) was preincubated at 37 ◦ C for 5 min in a 0.1 M potassium phosphate buffer, pH 7.50, containing 1 mM EDTA, 5 mM glucose-6-phosphate, 0.5 mM NADPH, 1 U/ml glucose-6-phosphate dehydrogenase, 2 U/ml catalase, 2 mM metyrapone, and 75 M thiocholine. The thiourea-containing compounds were added dissolved in methanol and samples were taken at fixed timepoints. In time, thiocholine is oxidized by the sulfenic acids formed by FMOs from the thiourea. The reaction remains linear in time until all thiocholine has been oxidized. The thiocholine remaining in the samples was determined spectrophotometrically with DTNB at 405 nm using a Packard Argus 400 microplate reader.
Fig. 1. Chemical structures and compound number of all thioureacontaining compounds used in this study. Compounds 1–4: alkylsubstituted thiourea. Compounds 5–12: aromatically-substituted thiourea.
Depletion of intracellular GSH content in precisioncut rat liver slices is shown in Fig. 3. ␣-Naphthylthiourea was found to cause a significant depletion of intracellular GSH after 2 h. Since ␣-naphthylthiourea did not cause any significant LDH leakage after 2 h (Fig. 2) it can be concluded that depletion of intracellular GSH indeed occurred. However, although 8 and 9
3. Results Chemical structures of the thiourea-containing compounds used in this study are shown in Fig. 1. As can be seen in Fig. 2, time-dependent cytotoxicity, measured as LDH leakage, could be observed in precision-cut rat liver slices. ␣-Naphthylthiourea was used as a positive control, since it has been shown to cause cytotoxicity in freshly prepared rat hepatocytes (Onderwater et al., 1998). A concentration of 1000 M of N-p-bromophenyl, N -(4-imidazole-ethyl)thiourea (8) or N-p-nitrophenyl, N -(4-imidazole-ethyl)thiourea (9) caused more than 95% LDH leakage in 6 h. Control levels of cell death (1% methanol only) were below 20% in 6 h. N-p-Methoxyphenyl, N -(4imidazole-ethyl)thiourea (6) caused 40 ± 19.7% LDH leakage in 6 h.
Fig. 2. Cytotoxicity towards precision-cut rat liver slices, evidenced as LDH leakage, caused by exposure to several thiourea-containing compounds. (䊐) Control (1% methanol); (䊏) ␣-naphthylthiourea; (䊉) N-p-methoxyphenyl, N -(4-imidazole-ethyl)thiourea; (䉱) N-pbromophenyl, N -(4-imidazole-ethyl)thiourea; (䊊) N-p-nitrophenyl, N -(4-imidazole-ethyl)thiourea. Compounds were added in a 1 mM concentration (n = 3 slices).
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Fig. 3. Intracellular GSH content of precision-cut rat liver slices after exposure to several thiourea-containing compounds. (䊐) Control (1% methanol); (䊏) ␣-naphthylthiourea; (䊉) N-pmethoxyphenyl, N -(4-imidazole-ethyl)thiourea; (䉱) N-p-bromophenyl, N -(4-imidazole-ethyl)thiourea; (䊊) N-p-nitrophenyl, N -(4-imidazole-ethyl)thiourea. Compounds were added in a 1 mM concentration (n = 3 slices). Values are expressed per mg of slice protein content.
also caused a significant reduction in intracellular GSH levels (Fig. 3), this reduction in intracellular GSH levels was mirrored by a reduction in intracellular LDH levels (Fig. 2). Therefore, depletion of intracellular GSH likely did not occur due to GSH oxidation or conjugation by metabolites of the thiourea-containing compound, but rather through GSH leakage from the cells due to loss of cell membrane integrity. Interestingly, intracellular GSH levels after 2 and 4 h of exposure to 9 appeared to be significantly higher than expected when compared to the percentage of LDH leakage at the same time points. MTT reduction activity of homogenates of precision-cut rat liver slices was tested as a possible marker for mitochondrial activity (Lomonte et al., 1993; Sawyer et al., 1994), since it has been proposed that the mechanism of cytotoxicity of the thiourea-containing insecticide diafenthiuron (3-(2,6diisopropyl-4-phenoxyphenyl)-1-tert-butyl-thiourea) involves inhibition of mitochondrial activity (Ruder and Kayser, 1992). The inhibition of mitochondrial activity is proposed to result from inhibition of mitochondrial ATP synthesis and covalent binding of the carbodiimide metabolite to the mitochondrial AT-
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Fig. 4. MTT reduction of slice homogenates after exposure of precision-cut rat liver slices to several thiourea-containing compounds. Values are expressed as percentages of the MTT reduction activity of homogenates of control slices (1% methanol) of the same incubation times. (䊉) N-p-Methoxyphenyl, N -(4-imidazole-ethyl)thiourea; (䉱) N-p-bromophenyl, N -(4-imidazole-ethyl)thiourea; (䊊) N-p-nitrophenyl, N -(4-imidazoleethyl)thiourea. Compounds were added in a 1 mM concentration (n = 3 slices).
Pase. The carbodiimide metabolite of diafenthiuron is formed by photolysis as well as oxidative metabolism of diafenthiuron (Petroske and Casida, 1995). MTT reduction activity of homogenates of precision-cut rat liver slices exposed to 1000 M 8 and 9 was significantly reduced after 2, 4, and 6 h. MTT reduction activity of slice homogenates of precision-cut rat liver slices exposed to 1000 M 6 was not reduced. Since MTT reduction activity of control slices (1% methanol only) showed a steady increase in time, up to 40% in 6 h, values in Fig. 4 are expressed as the percentage of MTT reduction activity of the respective control slices of the same time point. The possible STR among the five N-phenyl-substituted N -(4-imidazole-ethyl)thiourea (5–9) (plus N-naphthyl, N -(4-imidazole-ethyl)thiourea (12)) found at 1000 M was investigated more thoroughly. In Fig. 5 cytotoxicity, evidenced as LDH leakage, towards precision-cut rat liver slices caused by a homologous series of N-phenyl substituted N -(4-imidazole-ethyl)thiourea after 6 h is shown. A structure-dependency in cytotoxicity can indeed be observed. N-phenyl, N -(4-imidazole-ethyl)thiourea
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Fig. 5. Cytotoxicity towards precision-cut rat liver slices evidenced as LDH leakage caused by a homologous series of N-substituted N -(4-imidazole-ethyl)thiourea after 6 h. All compounds were added in concentrations of 0, 100, 200, and 500 M (average of two independent experiments with three slices).
(5) and 6 were found to be non-toxic up to a concentration of 500 M. Compound 12 was found to cause significant cytotoxicity at a concentration of 500 M. Compounds N-p-chlorophenyl, N -(4-imidazole-ethyl)thiourea (7) and 8 were already found to cause significant cytotoxicity at a concentration of 200 M. Compound 9 was also found to cause significant cytotoxicity at a concentration of 100 M and was found to cause significantly more cytotoxicity at 200 M than 7 and 8. LDH leakage from
precision-cut rat liver slices exposed to N-substituted N -(4-imidazole-ethyl)thiourea was mirrored by a simultaneous decrease in intracellular GSH levels. In Fig. 6, the levels of intracellular GSH levels of rat liver slices after 6 h of exposure to 7, 8, and 9 is shown. Intracellular GSH levels were significantly decreased after 6 h of exposure to 200 M of compound 9. Intracellular GSH levels were significantly decreased after 6 h of exposure to 500 M of 7, 8, or 9. Compounds 5 and 6 were shown not to decrease
Fig. 6. GSH depletion in precision-cut rat liver slices caused by a homologous series of N-substituted N -(4-imidazole-ethyl)thiourea after 6 h. All compounds were added in concentrations of 0, 100, 200, and 500 M (average of two independent experiments with three slices). Values are expressed per mg of slice protein content.
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4. Discussion
Fig. 7. Lineweaver–Burk plots of N-phenyl, N -(4-imidazole-ethyl) thiourea-dependent oxidation of thiocholine. (䊐) N-Phenyl, N (4-imidazole-ethyl)thiourea; (䊏) N-p-chloroyphenyl, N -(4-imidazole-ethyl)thiourea; (䊉) N-p-methoxyphenyl, N -(4-imidazoleethyl)thiourea; (䉱) N-p-bromophenyl, N -(4-imidazole-ethyl)thiourea; (䊊) N-p-nitrophenyl, N -(4-imidazole-ethyl)thiourea.
intracellular GSH levels as compared to control levels. An attempt was made to correlate the structuredependent cytotoxicity of N-substituted N -(4-imidazole-ethyl)thiourea to the enzyme kinetic parameters for the bioactivation of the thiourea-moiety by hepatic FMOs. Thiourea-containing compounds are bioactivated by FMOs to highly reactive sulfenic acids. Formation of sulfenic acids from thioureacontaining compounds can be determined using thiocholine as a probe thiol. Km and Vmax /Km values for thiourea-dependent oxidation of thiocholine of all thiourea-containing compounds tested for cytotoxicity in this study are presented in Table 2. Highest Vmax /Km values (in decreasing order) were found for ␣-naphthylthiourea, 9, 8, 12, and 7. In Fig. 7, Lineweaver–Burk plots for the formation of the thiol-reactive sulfenic acid metabolite by hepatic FMO enzymes from p-substituted N-phenyl, N -(4imidazole-ethyl)thiourea is shown. All plots intersect at the Y-axis indicating that Vmax values for the psubstituted N-phenyl, N -(4-imidazole-ethyl)thiourea are identical. Furthermore, large differences in slopes can be observed which translate into large differences in Km values.
The aim of this study was to compare the cytotoxicity of a series of thiourea-containing compounds in precision-cut rat liver slices in order to derive structure–toxicity relationships. STRs can be used in order to achieve a more rational design of thiourea-containing drugs and drug candidates, such as thiourea-containing histamine H3 receptor antagonists and non-nucleoside HIV reverse transcriptase inhibitors. Precision-cut rat liver slices were found to be an excellent tool to investigate STRs of thiourea-containing compounds. The viability of control slices remained high during 6 h of incubation, with less than 20% cell death, evidenced as LDH leakage. A time-dependent increase in cytotoxicity was observed with 1000 M of ␣-naphthylthiourea, ultimately reaching 99.4 ± 11.0% cytotoxicity after 6 h. In Table 1, it is shown that a concentration of 1000 M of N-p-bromophenyl, N -(4-imidazole-ethyl)thiourea (8) or 1000 M of N-p-nitrophenyl, N -(4-imidazole-ethyl)thiourea (9) caused more than 95% LDH leakage in 6 h, but N-p-methoxyphenyl, N -(4-imidazole-ethyl)thiourea (6) caused only 40% LDH leakage in 6 h. The other compounds were found not to be cytotoxic at 1000 M. This structure–toxicity relationship was further investigated by incubating different concentrations of N-p-phenyl-substituted N -(4-imidazoleethyl)thiourea. The results, shown in Fig. 5, indicate Table 1 Cytotoxicity of 1000 M of N-(4-imidazole-ethyl)thioureacontaining compounds towards precision-cut rat liver slices Compound no.
LDH leakage (%)
1 2 3 4 5 6 7 8 9 10 11 12 ␣-Naphthylthiourea
1.5 5.6 1.5 14.3 7.2 40.6 38.7 98.6 98.3 9.0 8.1 109.6 99.4
Effects after 6 h are reported (n = 3).
± ± ± ± ± ± ± ± ± ± ± ± ±
0.0 1.4 0.0 3.0 0.3 19.7 2.5 0.2 0.5 1.7 4.5 24.7 11.0
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that the cytotoxicity of 9 > 8 = N-p-chlorophenyl, N -(4-imidazole-ethyl)thiourea (7) > 6 > N-phenyl, N -(4-imidazole-ethyl)-thiourea (5). Furthermore, N␣-naphthyl, N -(4-imidazole-ethyl)thiourea (12) was found to be cytotoxic towards precision-cut rat liver slices during 6 h of incubation at a concentration of 500 M. Cytotoxicity towards precision-cut rat liver slices was accompanied by a simultaneous decrease in intracellular GSH levels, but was not as clearly preceded by a decrease in intracellular GSH as was also shown to occur in a previous study (Onderwater et al., 1998). In the latter study, exposure of freshly prepared rat hepatocytes to several mono- and di-substituted thiourea-containing compounds resulted in a decrease in intracellular GSH which preceded cell death. In the present study, this appeared to occur in the case of ␣-naphthylthiourea, in which case 2 h of exposure to 1000 M caused a depletion of 60% of intracellular GSH with less than 10% of cytotoxicity, but not in the case of the N-substituted N -(4-imidazole-ethyl)thiourea. Therefore, the GSH depletion determined in the present study might be the result of GSH leakage rather than oxidation of conjugation by metabolites of the thiourea-containing compound. MTT reduction activity of homogenates of the precision-cut rat liver slices was found to decrease in time after exposure to cytotoxic N-substituted N (4-imidazole-ethyl)thiourea a (Fig. 4). This activity was determined as a marked for the mitochondrial activity, and therefore, viability of precisioncut rat liver slices (Sawyer et al., 1994). However, since the decrease in MTT reduction activity accompanied, but not preceded cytotoxicity, it is likely that the decrease in MTT reduction activity is the result of leakage of pyridine nucleotides from the cells rather than a decrease in mitochondrial activity. Pyridine nucleotide-dependent mitochondrial MTT reduction was shown to account for up to 90% of total cellular MTT reduction activity (Berridge and Tan, 1993). Therefore, the decrease in MTT reduction activity of the slice homogenates resulting from exposure of the slices to thiourea-containing compounds is likely not to reflect a toxic effect towards mitochondria which is the proposed mechanism of cytotoxicity of the carbodiimide breakdown product and metabolite of the
thiourea-containing insecticide diafenthiuron (3-(2,6diisopropyl-4-phenoxyphenyl)-1-tert-butyl-thiourea) (Ruder and Kayser, 1992; Petroske and Casida, 1995). The N-alkyl- and N-alkylphenyl-substituted N -(4imidazole-ethyl)thiourea were found to be non-toxic towards precision-cut rat liver slices when exposed for 6 h to a concentration of 1000 M. It is believed that FMO enzymes play a crucial role in the bioactivation of thiourea-containing compounds (Poulsen et al., 1979; Guo and Ziegler, 1991; Guo et al., 1992; Smith and Crespi, 2002). Thiolreactive sulfenic acids are thought to be formed from the thiourea-moiety which cause a depletion of intracellular GSH and subsequently alkylate protein thiols. It has been shown that cytotoxicity of thiourea-containing compounds towards freshly prepared rat hepatocytes is accompanied by a depletion in intracellular GSH (Onderwater et al., 1998). This depletion of intracellular GSH can be the result of a futile cycle of sulfenic acid formation from the thiourea-containing compound by FMO, followed by formation of a thiourea–GSH mixed disulfide, which due to disulfide exchange ultimately leads to the formation of a GSH disulfide and the regeneration of the original thiourea-containing compound. GSH depletion can also be the result of the formation of a stable thiourea–GSH adduct which was shown to be formed in the case of N-(5-chloro-2-methylphenyl) N -(2-methylpropyl)thiourea by Stevens et al. (1997). It has also been shown that thiourea-containing compounds specifically alkylate protein thiols after bioactivation by hepatic FMO enzymes (Onderwater et al., 1999). In the latter study, a relationship was found between alkylating potential of thiourea-containing compounds towards the microsomal glutathione-5transferase, which shows increased activity after alkylation of cysteine-49, and Km value for the formation of sulfenic acids from the thiourea-moiety by hepatic FMO enzymes (Onderwater et al., 1999). In the present study, Km and Vmax /Km values for the formation of sulfenic acids from N-substituted N -(4imidazole-ethyl)thiourea by hepatic FMO were also determined. Large differences in Km and Vmax /Km values were found (Table 2), ranging from a Km value of 1011 ± 372 M and a Vmax /Km value of 0.00943 ± 0.00049 ml/(min mg) for 3 to a Km value of 20±11 M and Vmax /Km value of 0.17464±0.07997 ml/(min mg)
R.C.A. Onderwater et al. / Toxicology 197 (2004) 81–91 Table 2 Thiourea-dependent oxidation of thiocholine (n = 3) Compound no.
Km (M)
1 2 3 4 5 6 7 8 9 10 11 12 ␣-Naphthylthiourea
761 664 1011 194 262 176 44 35 20 463 301 29 3.0
± ± ± ± ± ± ± ± ± ± ± ± ±
171 35 372 21 31 57 5 10 11 46 126 4 0.3
Vmax /Km (ml/(min kg)) 0.01342 0.00676 0.00943 0.01595 0.01539 0.01971 0.07880 0.10830 0.17464 0.00675 0.00428 0.09615 0.54
± ± ± ± ± ± ± ± ± ± ± ± ±
0.00072 0.00014 0.00049 0.00110 0.00063 0.00310 0.00662 0.02341 0.07997 0.00024 0.00081 0.00629 0.02
Determined in rat liver microsomes according to Guo and Ziegler (1991).
for 9. Among a homologous series max of N-p-phenylsubstituted N -(4-imidazole-ethyl)thiourea Vmax /Km values could be ranked in the following order: 9 > 8 > 7 > 6 > 5. Km and Vmax /Km values of 12 were found to be similar to those for 8. The Km and Vmax /Km values of N-alkyl- and N-alkylphenyl-substituted N -(4-imidazole-ethyl)thiourea were found to be similar to or even higher than those for the non-cytotoxic compound 5. In Fig. 7, Lineweaver–Burk plots for the formation of the thiol-reactive sulfenic acid metabolite by hepatic FMO enzymes from p-substituted N-phenyl, N (4-imidazole-ethyl)thiourea are shown. As can be seen, all plots intersect at the Y-axis indicating that Vmax values for the p-substituted N-phenyl, N -(4imidazole-ethyl)thiourea are identical, which is expected from the unusual catalytic cycle of the FMO enzyme (Ziegler, 1993). From Tables 1 and 2 and Fig. 5 it can be concluded that a correlation exists between cytotoxicity towards precision-cut rat liver slices and Vmax /Km values for the formation of sulfenic acids from N-substituted N -(4-imidazole-ethyl)thiourea. The compound with the highest Vmax /Km value for the formation of sulfenic acids, 9, was also the most cytotoxic. Compounds with a significantly lower Vmax /Km value for the formation of sulfenic acids, 12, 8, and 7, were less cytotoxic than 9. Compounds with a Vmax /Km value for the formation of sulfenic acids lower than 0.0788 ml/(min mg) were found to be non-cytotoxic
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towards precision-cut rat liver slices for concentrations up to 1000 M and an exposure of 6 h. A similar correlation between cytotoxicity and Vmax /Km value for the formation of sulfenic acids by hepatic FMO was not found for mono- and di-substituted thiourea-containing compounds in freshly prepared rat hepatocytes (Onderwater et al., 1998). However, Km values for hepatic FMO of the thiourea-containing compounds used in this study were much lower. Therefore, differences in stability and the resultant reactivity of the reactive intermediates formed were proposed to be the determinant of STR found. These differences in stability and reactivity might also be a possible explanation for the STR found in the present study. Davis et al. (1985) showed that the high reactivity of benzenesulfenic acids can be reduced by introducing the possibility of intramolecular hydrogen bonding of the sulfenic acid moiety and by reducing the electron density of the sulfenic acid sulfur atom by introducing electron-withdrawing substituents to the benzene ring at the ortho- or para-positions. Decker et al. (1992) suggested that the potential for intramolecular hydrogen bonding to be a determining factor in the stability of the sulfenic acids formed as initial metabolites from a series of thiourea moiety-containing benzimidazoline-2thiones. In conclusion, this study has shown that N-phenyl substituted N -(4-imidazole-ethyl)thiourea-containing electron-withdrawing p-substituents are cytotoxic towards precision-cut rat liver slices. Cytotoxicity is increased with increasing electron withdrawing capacity of the p-substituent. A correlation exists between Vmax /Km value for the formation of sulfenic acids by rat liver FMO enzymes and cytotoxicity.
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M.D., Lind, P., Morin Jr., J.M., Muesing, M.A., Noreén, R., Öberg, B., Pranc, P., Sahlberg, C., Ternansky, R.J., Vasileff, R.T., Vrang, L., West, S.J., Zhang, H., 1996. Phenethylthiazolylthiourea (PETT) compounds as a new class of HIV-1 reverse transcriptase inhibitors. 2. Synthesis and further structure–activity relationship studies of PETT analogs. J. Med. Chem. 39, 4261–4274. Davis, F.A., Jenkins, L.A., Billmers, R.L., 1985. Chemistry of sulfenic acids. 7. Reason for the high reactivity of sulfenic acids. Stabilization by intramolecular hydrogen bonding and electronegative effects. J. Org. Chem. 51, 1033– 1040. D’Cruz, O.J., Venkatachalam, T.K., Mao, C., Qazi, S., Uckun, F.M., 2002. Structural requirements for potent antihuman immunodificiency virus (HIV) and sperm-immobilizing activities of cyclohexenyl thiourea and urea non-nucleoside inhibitors of HIV-1 reverse transcriptase. Biol. Reprod. 67, 1959–1974. D’Cruz, O.J., Dong, Y., Uckun, F.M., 2003. Potent dual anti-HIV and spermicidal activities of novel oxovanadium(V) complexes with thiourea non-nucleoside inhibitors of HIV-1 reverse transcriptase. Biochem. Biophys. Res. Commun. 302, 253– 264. Decker, C.J., Doerge, D.R., Cashman, J.R., 1992. Metabolism of benzimidazoline-2-thiones by rat hepatic microsomes and hog liver flavin-containing monooxygenase. Chem. Res. Toxicol. 5, 726–733. Forrest, J.A.H., Shearman, D.J.C., Spence, R., Celestin, L.R., 1975. Neutropenia associated with metiamide. Lancet, I, 392– 393. Guo, W.-X.A., Ziegler, D.M., 1991. Estimation of flavin-containing monooxygenase activities in crude tissue preparations by thiourea dependent oxidation of thiocholine. Anal. Biochem. 198, 143–148. Guo, W.-X.A., Poulsen, L.L., Ziegler, D.M., 1992. Use of thiocarbamides as selective substrate probes for isoforms of flavin-containing monooxygenases. Biochem. Pharmacol. 44, 2029–2037. Högberg, M., Morrison, I., 2000. HIV-1 non-nucleoside reverse transcriptase inhibitors. Exp. Opin. Ther. Patents 10, 1189– 1199. Lomonte, B., Gutiérrez, J.M., Romero, M., Núñez, J., Tarkowski, A., Hanson, L.Å., 1993. An MTT-based method for the in vivo quantification of myotoxic activity of snake venoms and its neutralization by antibodies. J. Immunol. Methods 161, 231– 237. Mao, C., Sudbeck, E.A., Venkatachalam, T.K., Uckun, F.M., 1999. Structure-based design of non-nucleoside reverse transcriptase inhibitors of drug-resistant human immunodeficiency virus. Antiviral Chem. Chemother. 10, 233–240. Onderwater, R.C.A., Commandeur, J.N.M., Groot, E.J., Sitters, A., Menge, W.M.P.B., Vermeulen, N.P.E., 1998. Cytotoxicity of a series of mono- and di-substituted thiourea in freshly isolated rat hepatocytes: a preliminary structure–toxicity relationship study. Toxicology 125, 117–129. Onderwater, R.C.A., Commandeur, J.N.M., Menge, W.M.P.B., Vermeulen, N.P.E., 1999. Activation of microsomal glutathione
S-transferase and inhibition of CYP1A1 activity as a model system to detect protein alkylation by thiourea-containing compounds in rat liver microsomes. Chem. Res. Toxicol. 12, 396–402. Pedersen, O.S., Pedersen, E.B., 1999. Non-nucleoside reverse transcriptase inhibitors: the NNRTI boom. Antiviral Chem. Chemother. 10, 285–314. Petroske, E., Casida, J.E., 1995. Diafenthiuron action: carbodiimide formation and ATPase inhibition. Pest. Biochem. Physiol. 53, 60–74. Poulsen, L.L., Hyslop, R.M., Ziegler, D.M., 1979. S-Oxygenation of N-substituted thioureas catalyzed by the pig liver microsomal FAD-containing monooxygenase. Arch. Biochem. Biophys. 198, 78–88. Redegeld, F.A.M., Van Opstal, M.A.J., Houdkamp, E., Van Bennekom, W.P., 1988. Determination of glutathione in biological material by flow-injection analysis using an enzymatic recycling reaction. Anal. Biochem. 174, 489–495. Ruder, F.J., Kayser, H., 1992. The carbodiimide product of diafenthiuron reacts covalently with two mitochondrial proteins, the F0-proteolipid and porin, and inhibits mitochondrial ATPase in vitro. Pest. Biochem. Physiol. 42, 248–261. Sawyer, J.S., Daller, J.A., Brendel, K., Yohem, K., Putnam, C.W., 1994. The hepatoxicities of endotoxin and ethanol: comparisons in vitro using the precision-cut rat slice model. Life Sci. 55, 1407–1417. Skellern, G.G., 1989. Thiocarbamides. In: Damani, L.A. (Ed.), Sulphur Containing Drugs and Related Organic Compounds. Chemistry, Biology, Toxicity, Part 1B. Ellis Horwood Ltd., Chichester, UK,p. 49. Smith, P.B., Crespi, C., 2002. Thiourea toxicity in mouse C3H/10T1/2 cells expressing human flavin dependent monooxygenase 3. Biochem. Pharmacol. 63, 1941–1948. Stevens, G.J., Hitchcock, K., Wang, Y.K., Coppola, G.M., Versace, R.W., Chin, J.A., Shapiro, M., Suwanrumpha, S., Mangold, B.L.K., 1997. In vitro metabolism of N-(5chloro-2-methylphenyl)-N -(2-methylpropyl)thiourea: species comparison and identification of a novel thiocarbamide– glutathione adduct. Chem. Res. Toxicol. 10, 733–741. Uckun, F.M., Mao, C., Pendergrass, S., Maher, D., Zhu, D., Tuel-Ahlgren, L., Venkatachalam, T.K., 2000. N-[2(4-Methylphenyl)ethyl]-N -[2-(5-bromopyridyl)]-thiourea as a potent inhibitor of NNRTI-resistant and multidrug-resistant human immunodeficiency virus type 1. Antiviral Chem. Chemother. 11, 135–140. Van der Goot, H., Schepers, M.J.P., Sterk, G.J., Timmerman, H., 1992. Isothiourea analogues of histamine as potent agonists or antagonists of the histamine H-3-receptor. Eur. J. Med. Chem. 27, 511–517. Van der Straat, R., De Vries, J., Debets, A.J.J., Vermeulen, N.P.E., 1987. The mechanism of prevention of paracetamolinduced hepatotoxicity by 3,5-dialkylsubstitution. The roles of glutathione depletion and oxidative stress. Biochem. Pharmacol. 36, 2065–2070. Venkatachalam, T.K., Sudbeck, E.A., Mao, C., Uckun, F.M., 2000. Stereochemistry of halopyridyl and thiazolyl thiourea compounds is a major determinant of their potency as
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Comparative cytotoxicity of N-substituted N-(4-imidazole-ethyl)thiourea in precision-cut rat liver slices Rob C.A. Onderwater, Jan N.M. Commandeur, Nico P.E. Vermeulen∗ Leiden/Amsterdam Center for Drug Research (LACDR), Division of Molecular Toxicology, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands Received in revised form 17 November 2003; accepted 17 November 2003
Abstract In order to more rationally design thiourea-containing drugs and drug candidates, specifically thiourea-containing histamine H3 receptor antagonists, it is necessary to develop structure–toxicity relationships (STRs). For this purpose, the cytotoxicity of a series of thiourea-containing compounds was tested in precision-cut rat liver slices. A concentration of 1000 M of N-p-bromophenyl, N -(4-imidazole-ethyl)thiourea (8) or N-p-nitrophenyl, N -(4-imidazole-ethyl)thiourea (9) was found to cause cytotoxicity, evidenced as LDH leakage, resulting in more than 95% LDH leakage after 6 h. N-p-Methoxyphenyl, N -(4-imidazole-ethyl)thiourea (6) caused 40.6 ± 19.7% LDH leakage after 6 h. Control levels of cell death (1% methanol as control vehicle) were below 20% in 6 h. After 6 h of exposure, N-p-chlorophenyl, N -(4-imidazole-ethyl)thiourea (7), 8, and 9 were already found to cause significant cytotoxicity at a concentration of 100 M. At 200 M, 9 was found to cause significantly more cytotoxicity than 7 and 8. N-Naphthyl, N -(4-imidazole-ethyl)thiourea (12) was found to cause significant cytotoxicity towards precision-cut rat liver slices after 6 h of exposure to a concentration of 500 M. All other N-substituted, N -(4-imidazole-ethyl)thiourea tested in this study were not found to be cytotoxic towards precision-cut rat liver slices within the 6 h of exposure up to a concentration of 1000 M. Intracellular glutathione (GSH) content and mitochondrial MTT reduction activity were also examined after exposure of slices to N-substituted, N -(4-imidazole-ethyl)thiourea. Both of these markers, however, were not found to provide additional information regarding the possible mechanisms of cytotoxicity, i.e. GSH depletion or reduced mitochondrial activity since these markers did not clearly precede LDH leakage. A correlation was found between cytotoxicity towards precision-cut rat liver slices and Vmax /Km values for the formation of sulfenic acids from N-substituted N -(4-imidazole-ethyl)thiourea by hepatic rat flavin-containing monooxygenases (FMO). The compound with the highest Vmax /Km value for the formation of sulfenic acids, 9, was also the most cytotoxic. Compounds with a significantly lower Vmax /Km value, 7, 8, and 12, were less cytotoxic than 9. Compounds with a Vmax /Km value for the formation of sulfenic acids lower than 0.0788 ml/(min mg) were found not to be cytotoxic towards precision-cut rat liver slices for concentrations up to 1000 M at an exposure time of 6 h. It is concluded, from this study, that N-phenyl substituted N -(4-imidazole-ethyl)thiourea-containing electron-withdrawing p-substituents are cytotoxic towards precision-cut rat liver slices. Cytotoxicity is increased with increasing electron-withdrawing
∗ Corresponding author. Tel.: +31-20-4447590; fax: +31-20-4447610. E-mail address: [email protected] (N.P.E. Vermeulen).
0300-483X/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2003.11.014
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capacity of the p-substituent. A correlation was found to exist between Vmax /Km value for the formation of sulfenic acids by rat liver FMO enzymes and cytotoxicity. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Comparative cytotoxicity; N -(4-Imidazole-ethyl)thiourea; Precision-cut rat liver slices
1. Introduction Interest in the thiourea group as a pharmacophore has rekindled with the development of some promising new thiourea-containing drugs candidates. Among these are potent HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTI). Trovirdine was the first thiourea-containing compound of this class to reach Phase I clinical trails. It was developed through rational design from a series of N-phenylethyl, N thiazolylthiourea (PETT) by Eli Lilly and Medivir (Cantrell et al., 1996; Pedersen and Pedersen, 1999; Högberg and Morrison, 2000). Using a computer model of HIV-1 reverse transcriptase thioureacontaining compounds were designed that show even better activity against the wild type reverse transcriptase as trovirdine and are specifically designed to also strongly inhibit the most often observed resistant strains (Mao et al., 1999; Uckun et al., 2000; Venkatachalam et al., 2000). Several of the newly designed PETT-class NNRTIs have also been shown to have spermicidal activity in the low micromolar range creating. This combination gives these compounds a unique clinical potential of becoming the active ingredients of a vaginal contraceptive for women who are at high risk for acquiring an HIV infection (D’Cruz et al., 2002, 2003). Another interesting lead for drug development can be found among centrally active histamine H3 antagonists (Vollinga et al., 1995). Many of the latter compounds are analogues of one of the first thiourea-containing drugs considered for widespread use, namely the histamine H2 receptor antagonist burimamide. However, its application was never a success, due to its low bioavailability as well as the severe side effects which accompanied its use (Forrest et al., 1975). Thiourea-containing compounds are often goitrogenic and inhibitory towards thyroid hormone biosynthesis, and some cause hypersensitivity reac-
tions and are pulmonary or hepatotoxins (Skellern, 1989). Most adverse reactions are attributed to the thiocarbonyl moiety since in many cases the corresponding urea compounds do not cause similar toxicity (Skellern, 1989; Onderwater et al., 1998). The majority of the studies into thiourea toxicity have been performed with the pulmonary toxin ␣naphthylthiourea. Early radioactivity studies in the 1970s with ␣-naphthylthiourea have shown that both the thionocarbon- and the thionosulfur-atom are covalently bound to rat lung microsomal proteins upon bioactivation (Boyd and Neal, 1976). Oxidation of the thionosulfur moiety is generally accepted to be the first step in thiourea bioactivation. It is generally believed that flavin-containing monooxygenases (FMOs) are responsible for this oxidation, which results in the formation of thiolreactive sulfenic acids (Poulsen et al., 1979; Guo and Ziegler, 1991; Guo et al., 1992; Smith and Crespi, 2002). In order to achieve a rational design of thioureacontaining drugs and drug candidates, more specifically thiourea-containing histamine H3 receptor antagonists, it is necessary to develop structure– toxicity relationships (STRs) of thiourea-containing compounds. For this purpose, the cytotoxicity of a series of 13 thiourea-containing compounds was tested in precision-cut rat liver slices. In order to develop STRs which can directly be applied to histamine antagonists, like burimamide, compounds were synthesized which closely resemble burimamide, without being pharmacologically active. Thus, the pharmacological activity of histamine antagonists was prevented from interfering with the occurrence of cytotoxity in precision-cut rat liver slices. Therefore, N-substituted N -(4-imidazole-ethyl)thiourea, short-chain analogues of burimamide (N-methyl, N (4-imidazole-butyl)thiourea) were used to develop STRs.
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2. Materials and methods 2.1. Chemicals 5,5 -Dithiobis(2-nitrobenzoic acid) (DTNB) was obtained from Fluka Chemie AG (Buchs, Switzerland). NADPH was obtained from Boehringer (Mannheim, Germany), N-ethylmaleimide (NEM) from Aldrich (Steinheim, Germany) and acetylthiocholine from Sigma (St. Louis, USA). N-substituted N -(4-imidazole-ethyl)thiourea were synthesized according to Van der Goot et al. (1992). All other chemicals were of the highest grade commercially available. 2.2. Preparation of precision-cut rat liver slices Rat liver slices were prepared using a Krumdieck Tissue Slicer (Alabama Research and Development). Livers were isolated from male Wistar rats (200– 250 g), which were obtained from Harlan (Zeist, The Netherlands) and which were fed a standard laboratory diet from Hope Farms (Woerden, The Netherlands) and had access to food and water ad libitum. Rats were sacrificed by decapitation and livers were quickly removed. Excised livers were stored at 4 ◦ C in Krebs–Henseleit buffer (pH 7.40) containing 2 g dsglucose/l, which was saturated with carbogen (95% O2 /5% CO2 ), (KH buffer) until cylindrical cores were made with a 5 mm coring tool (Alabama Research and Development). Slices of 250–300 m were made at 4 ◦ C in KH buffer within 30 min of liver isolation. 2.3. Incubation of precision-cut rat liver slices Slices were transferred, in sets of five, to 20 ml polypropylene scintillation vials, containing 3 ml ice-cold KH buffer. Vials were saturated with carbogen and, subsequently, were placed in a rotating incubation block set at 37 ◦ C. Slices were, thus, pre-incubated for 60 mm at 100 rotations per minute. After pre-incubation, single slices were transferred to 20 ml polypropylene scintillation vials, containing 1.5 ml KH buffer of 37 ◦ C. Vials were saturated with carbogen and were subsequently placed in an incubation block set at 37 ◦ C. Test compounds, dissolved in 50% 0.1 M potassium phosphate buffer (pH 7.40)/50% methanol, were added to the vials prior to addition of the slice. After addition of the compounds all incubations contained 1% methanol.
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The carbogen atmosphere was refreshed every 30 min throughout the experiments. At specific timepoints, rat liver slices were transferred from the incubation to Eppendorf vials containing 1 ml ice-cold phosphate buffered saline (PBS) and samples of 1 ml were taken from the incubation medium. Slices were homogenized at 4 ◦ C in a Potter–Ehlvejem tube. A sample of the slice homogenate was taken for protein determination, which was determined with the BIORAD protein reagent, using BSA as a standard, on a Packard Argus 400 microplate reader. 2.4. LDH leakage from precision-cut rat liver slices LDH leakage was determined in both incubation media and slice homogenates by measuring oxidation of NADH in the presence of sodium pyruvate at 340 nm using a Phillips PU-8720 UV-Vis spectrophotometer (Van der Straat et al., 1987). The final reaction mixture (volume 1 ml) contained 0.2 mM NADH and 0.5 mM sodium pyruvate in 50 mM triethanolamine buffer, pH 7.4. Addition of 100 l of the samples initiated the enzymic reaction. LDH leakage from the rat liver slices was calculated from the activities found in total incubation media and slice homogenates. 2.5. Intracellular GSH levels in precision-cut rat liver slices Total glutathione concentrations (GSH and GSSG) in both incubation media and slice homogenates were determined, using a Packard Argus 400 microplate reader, via an enzymatic recycling reaction of GSH in combination with a chromogenic reaction of DTNB, continuously leading to the formation of 5-thio-2-nitrobenzoate which were measured at 405 nm (Redegeld et al., 1988). Oxidized glutathione (GSSG) was determined analogously to the total glutathione measurement, except that GSH was first depleted through reaction with an excess N-ethylmale¨ımide. GSH levels were deduced from total glutathione minus GSSG levels. 2.6. MTT reduction by precision-cut rat liver slices MTT reduction activity was determined according to Lomonte et al. (1993). Slice homogenates were centrifuged for 10 min at 1000 × g. To 250 l of the
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supernatant 750 l of PBS and 200 l of 2.5 mg/ml MTT in PBS were added. The mixture was incubated in polypropylene vials in the dark for 90 min at 37 ◦ C. The yellow colored MTT is reduced to a purple formazan. The difference in absorbance at 570 and 690 nm was determined. 2.7. Thiourea-dependent oxidation of thiocholine by rat liver FMO Thiocholine was prepared from acetylthiocholine in acidic methanol according to Guo and Ziegler (1991). Thiourea-dependent oxidation of thiocholine was determined according to Guo and Ziegler (1991). Rat liver microsomes were prepared as described by Van der Straat et al. (1987). Microsomal protein (0.5 mg) was preincubated at 37 ◦ C for 5 min in a 0.1 M potassium phosphate buffer, pH 7.50, containing 1 mM EDTA, 5 mM glucose-6-phosphate, 0.5 mM NADPH, 1 U/ml glucose-6-phosphate dehydrogenase, 2 U/ml catalase, 2 mM metyrapone, and 75 M thiocholine. The thiourea-containing compounds were added dissolved in methanol and samples were taken at fixed timepoints. In time, thiocholine is oxidized by the sulfenic acids formed by FMOs from the thiourea. The reaction remains linear in time until all thiocholine has been oxidized. The thiocholine remaining in the samples was determined spectrophotometrically with DTNB at 405 nm using a Packard Argus 400 microplate reader.
Fig. 1. Chemical structures and compound number of all thioureacontaining compounds used in this study. Compounds 1–4: alkylsubstituted thiourea. Compounds 5–12: aromatically-substituted thiourea.
Depletion of intracellular GSH content in precisioncut rat liver slices is shown in Fig. 3. ␣-Naphthylthiourea was found to cause a significant depletion of intracellular GSH after 2 h. Since ␣-naphthylthiourea did not cause any significant LDH leakage after 2 h (Fig. 2) it can be concluded that depletion of intracellular GSH indeed occurred. However, although 8 and 9
3. Results Chemical structures of the thiourea-containing compounds used in this study are shown in Fig. 1. As can be seen in Fig. 2, time-dependent cytotoxicity, measured as LDH leakage, could be observed in precision-cut rat liver slices. ␣-Naphthylthiourea was used as a positive control, since it has been shown to cause cytotoxicity in freshly prepared rat hepatocytes (Onderwater et al., 1998). A concentration of 1000 M of N-p-bromophenyl, N -(4-imidazole-ethyl)thiourea (8) or N-p-nitrophenyl, N -(4-imidazole-ethyl)thiourea (9) caused more than 95% LDH leakage in 6 h. Control levels of cell death (1% methanol only) were below 20% in 6 h. N-p-Methoxyphenyl, N -(4imidazole-ethyl)thiourea (6) caused 40 ± 19.7% LDH leakage in 6 h.
Fig. 2. Cytotoxicity towards precision-cut rat liver slices, evidenced as LDH leakage, caused by exposure to several thiourea-containing compounds. (䊐) Control (1% methanol); (䊏) ␣-naphthylthiourea; (䊉) N-p-methoxyphenyl, N -(4-imidazole-ethyl)thiourea; (䉱) N-pbromophenyl, N -(4-imidazole-ethyl)thiourea; (䊊) N-p-nitrophenyl, N -(4-imidazole-ethyl)thiourea. Compounds were added in a 1 mM concentration (n = 3 slices).
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Fig. 3. Intracellular GSH content of precision-cut rat liver slices after exposure to several thiourea-containing compounds. (䊐) Control (1% methanol); (䊏) ␣-naphthylthiourea; (䊉) N-pmethoxyphenyl, N -(4-imidazole-ethyl)thiourea; (䉱) N-p-bromophenyl, N -(4-imidazole-ethyl)thiourea; (䊊) N-p-nitrophenyl, N -(4-imidazole-ethyl)thiourea. Compounds were added in a 1 mM concentration (n = 3 slices). Values are expressed per mg of slice protein content.
also caused a significant reduction in intracellular GSH levels (Fig. 3), this reduction in intracellular GSH levels was mirrored by a reduction in intracellular LDH levels (Fig. 2). Therefore, depletion of intracellular GSH likely did not occur due to GSH oxidation or conjugation by metabolites of the thiourea-containing compound, but rather through GSH leakage from the cells due to loss of cell membrane integrity. Interestingly, intracellular GSH levels after 2 and 4 h of exposure to 9 appeared to be significantly higher than expected when compared to the percentage of LDH leakage at the same time points. MTT reduction activity of homogenates of precision-cut rat liver slices was tested as a possible marker for mitochondrial activity (Lomonte et al., 1993; Sawyer et al., 1994), since it has been proposed that the mechanism of cytotoxicity of the thiourea-containing insecticide diafenthiuron (3-(2,6diisopropyl-4-phenoxyphenyl)-1-tert-butyl-thiourea) involves inhibition of mitochondrial activity (Ruder and Kayser, 1992). The inhibition of mitochondrial activity is proposed to result from inhibition of mitochondrial ATP synthesis and covalent binding of the carbodiimide metabolite to the mitochondrial AT-
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Fig. 4. MTT reduction of slice homogenates after exposure of precision-cut rat liver slices to several thiourea-containing compounds. Values are expressed as percentages of the MTT reduction activity of homogenates of control slices (1% methanol) of the same incubation times. (䊉) N-p-Methoxyphenyl, N -(4-imidazole-ethyl)thiourea; (䉱) N-p-bromophenyl, N -(4-imidazole-ethyl)thiourea; (䊊) N-p-nitrophenyl, N -(4-imidazoleethyl)thiourea. Compounds were added in a 1 mM concentration (n = 3 slices).
Pase. The carbodiimide metabolite of diafenthiuron is formed by photolysis as well as oxidative metabolism of diafenthiuron (Petroske and Casida, 1995). MTT reduction activity of homogenates of precision-cut rat liver slices exposed to 1000 M 8 and 9 was significantly reduced after 2, 4, and 6 h. MTT reduction activity of slice homogenates of precision-cut rat liver slices exposed to 1000 M 6 was not reduced. Since MTT reduction activity of control slices (1% methanol only) showed a steady increase in time, up to 40% in 6 h, values in Fig. 4 are expressed as the percentage of MTT reduction activity of the respective control slices of the same time point. The possible STR among the five N-phenyl-substituted N -(4-imidazole-ethyl)thiourea (5–9) (plus N-naphthyl, N -(4-imidazole-ethyl)thiourea (12)) found at 1000 M was investigated more thoroughly. In Fig. 5 cytotoxicity, evidenced as LDH leakage, towards precision-cut rat liver slices caused by a homologous series of N-phenyl substituted N -(4-imidazole-ethyl)thiourea after 6 h is shown. A structure-dependency in cytotoxicity can indeed be observed. N-phenyl, N -(4-imidazole-ethyl)thiourea
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Fig. 5. Cytotoxicity towards precision-cut rat liver slices evidenced as LDH leakage caused by a homologous series of N-substituted N -(4-imidazole-ethyl)thiourea after 6 h. All compounds were added in concentrations of 0, 100, 200, and 500 M (average of two independent experiments with three slices).
(5) and 6 were found to be non-toxic up to a concentration of 500 M. Compound 12 was found to cause significant cytotoxicity at a concentration of 500 M. Compounds N-p-chlorophenyl, N -(4-imidazole-ethyl)thiourea (7) and 8 were already found to cause significant cytotoxicity at a concentration of 200 M. Compound 9 was also found to cause significant cytotoxicity at a concentration of 100 M and was found to cause significantly more cytotoxicity at 200 M than 7 and 8. LDH leakage from
precision-cut rat liver slices exposed to N-substituted N -(4-imidazole-ethyl)thiourea was mirrored by a simultaneous decrease in intracellular GSH levels. In Fig. 6, the levels of intracellular GSH levels of rat liver slices after 6 h of exposure to 7, 8, and 9 is shown. Intracellular GSH levels were significantly decreased after 6 h of exposure to 200 M of compound 9. Intracellular GSH levels were significantly decreased after 6 h of exposure to 500 M of 7, 8, or 9. Compounds 5 and 6 were shown not to decrease
Fig. 6. GSH depletion in precision-cut rat liver slices caused by a homologous series of N-substituted N -(4-imidazole-ethyl)thiourea after 6 h. All compounds were added in concentrations of 0, 100, 200, and 500 M (average of two independent experiments with three slices). Values are expressed per mg of slice protein content.
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4. Discussion
Fig. 7. Lineweaver–Burk plots of N-phenyl, N -(4-imidazole-ethyl) thiourea-dependent oxidation of thiocholine. (䊐) N-Phenyl, N (4-imidazole-ethyl)thiourea; (䊏) N-p-chloroyphenyl, N -(4-imidazole-ethyl)thiourea; (䊉) N-p-methoxyphenyl, N -(4-imidazoleethyl)thiourea; (䉱) N-p-bromophenyl, N -(4-imidazole-ethyl)thiourea; (䊊) N-p-nitrophenyl, N -(4-imidazole-ethyl)thiourea.
intracellular GSH levels as compared to control levels. An attempt was made to correlate the structuredependent cytotoxicity of N-substituted N -(4-imidazole-ethyl)thiourea to the enzyme kinetic parameters for the bioactivation of the thiourea-moiety by hepatic FMOs. Thiourea-containing compounds are bioactivated by FMOs to highly reactive sulfenic acids. Formation of sulfenic acids from thioureacontaining compounds can be determined using thiocholine as a probe thiol. Km and Vmax /Km values for thiourea-dependent oxidation of thiocholine of all thiourea-containing compounds tested for cytotoxicity in this study are presented in Table 2. Highest Vmax /Km values (in decreasing order) were found for ␣-naphthylthiourea, 9, 8, 12, and 7. In Fig. 7, Lineweaver–Burk plots for the formation of the thiol-reactive sulfenic acid metabolite by hepatic FMO enzymes from p-substituted N-phenyl, N -(4imidazole-ethyl)thiourea is shown. All plots intersect at the Y-axis indicating that Vmax values for the psubstituted N-phenyl, N -(4-imidazole-ethyl)thiourea are identical. Furthermore, large differences in slopes can be observed which translate into large differences in Km values.
The aim of this study was to compare the cytotoxicity of a series of thiourea-containing compounds in precision-cut rat liver slices in order to derive structure–toxicity relationships. STRs can be used in order to achieve a more rational design of thiourea-containing drugs and drug candidates, such as thiourea-containing histamine H3 receptor antagonists and non-nucleoside HIV reverse transcriptase inhibitors. Precision-cut rat liver slices were found to be an excellent tool to investigate STRs of thiourea-containing compounds. The viability of control slices remained high during 6 h of incubation, with less than 20% cell death, evidenced as LDH leakage. A time-dependent increase in cytotoxicity was observed with 1000 M of ␣-naphthylthiourea, ultimately reaching 99.4 ± 11.0% cytotoxicity after 6 h. In Table 1, it is shown that a concentration of 1000 M of N-p-bromophenyl, N -(4-imidazole-ethyl)thiourea (8) or 1000 M of N-p-nitrophenyl, N -(4-imidazole-ethyl)thiourea (9) caused more than 95% LDH leakage in 6 h, but N-p-methoxyphenyl, N -(4-imidazole-ethyl)thiourea (6) caused only 40% LDH leakage in 6 h. The other compounds were found not to be cytotoxic at 1000 M. This structure–toxicity relationship was further investigated by incubating different concentrations of N-p-phenyl-substituted N -(4-imidazoleethyl)thiourea. The results, shown in Fig. 5, indicate Table 1 Cytotoxicity of 1000 M of N-(4-imidazole-ethyl)thioureacontaining compounds towards precision-cut rat liver slices Compound no.
LDH leakage (%)
1 2 3 4 5 6 7 8 9 10 11 12 ␣-Naphthylthiourea
1.5 5.6 1.5 14.3 7.2 40.6 38.7 98.6 98.3 9.0 8.1 109.6 99.4
Effects after 6 h are reported (n = 3).
± ± ± ± ± ± ± ± ± ± ± ± ±
0.0 1.4 0.0 3.0 0.3 19.7 2.5 0.2 0.5 1.7 4.5 24.7 11.0
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that the cytotoxicity of 9 > 8 = N-p-chlorophenyl, N -(4-imidazole-ethyl)thiourea (7) > 6 > N-phenyl, N -(4-imidazole-ethyl)-thiourea (5). Furthermore, N␣-naphthyl, N -(4-imidazole-ethyl)thiourea (12) was found to be cytotoxic towards precision-cut rat liver slices during 6 h of incubation at a concentration of 500 M. Cytotoxicity towards precision-cut rat liver slices was accompanied by a simultaneous decrease in intracellular GSH levels, but was not as clearly preceded by a decrease in intracellular GSH as was also shown to occur in a previous study (Onderwater et al., 1998). In the latter study, exposure of freshly prepared rat hepatocytes to several mono- and di-substituted thiourea-containing compounds resulted in a decrease in intracellular GSH which preceded cell death. In the present study, this appeared to occur in the case of ␣-naphthylthiourea, in which case 2 h of exposure to 1000 M caused a depletion of 60% of intracellular GSH with less than 10% of cytotoxicity, but not in the case of the N-substituted N -(4-imidazole-ethyl)thiourea. Therefore, the GSH depletion determined in the present study might be the result of GSH leakage rather than oxidation of conjugation by metabolites of the thiourea-containing compound. MTT reduction activity of homogenates of the precision-cut rat liver slices was found to decrease in time after exposure to cytotoxic N-substituted N (4-imidazole-ethyl)thiourea a (Fig. 4). This activity was determined as a marked for the mitochondrial activity, and therefore, viability of precisioncut rat liver slices (Sawyer et al., 1994). However, since the decrease in MTT reduction activity accompanied, but not preceded cytotoxicity, it is likely that the decrease in MTT reduction activity is the result of leakage of pyridine nucleotides from the cells rather than a decrease in mitochondrial activity. Pyridine nucleotide-dependent mitochondrial MTT reduction was shown to account for up to 90% of total cellular MTT reduction activity (Berridge and Tan, 1993). Therefore, the decrease in MTT reduction activity of the slice homogenates resulting from exposure of the slices to thiourea-containing compounds is likely not to reflect a toxic effect towards mitochondria which is the proposed mechanism of cytotoxicity of the carbodiimide breakdown product and metabolite of the
thiourea-containing insecticide diafenthiuron (3-(2,6diisopropyl-4-phenoxyphenyl)-1-tert-butyl-thiourea) (Ruder and Kayser, 1992; Petroske and Casida, 1995). The N-alkyl- and N-alkylphenyl-substituted N -(4imidazole-ethyl)thiourea were found to be non-toxic towards precision-cut rat liver slices when exposed for 6 h to a concentration of 1000 M. It is believed that FMO enzymes play a crucial role in the bioactivation of thiourea-containing compounds (Poulsen et al., 1979; Guo and Ziegler, 1991; Guo et al., 1992; Smith and Crespi, 2002). Thiolreactive sulfenic acids are thought to be formed from the thiourea-moiety which cause a depletion of intracellular GSH and subsequently alkylate protein thiols. It has been shown that cytotoxicity of thiourea-containing compounds towards freshly prepared rat hepatocytes is accompanied by a depletion in intracellular GSH (Onderwater et al., 1998). This depletion of intracellular GSH can be the result of a futile cycle of sulfenic acid formation from the thiourea-containing compound by FMO, followed by formation of a thiourea–GSH mixed disulfide, which due to disulfide exchange ultimately leads to the formation of a GSH disulfide and the regeneration of the original thiourea-containing compound. GSH depletion can also be the result of the formation of a stable thiourea–GSH adduct which was shown to be formed in the case of N-(5-chloro-2-methylphenyl) N -(2-methylpropyl)thiourea by Stevens et al. (1997). It has also been shown that thiourea-containing compounds specifically alkylate protein thiols after bioactivation by hepatic FMO enzymes (Onderwater et al., 1999). In the latter study, a relationship was found between alkylating potential of thiourea-containing compounds towards the microsomal glutathione-5transferase, which shows increased activity after alkylation of cysteine-49, and Km value for the formation of sulfenic acids from the thiourea-moiety by hepatic FMO enzymes (Onderwater et al., 1999). In the present study, Km and Vmax /Km values for the formation of sulfenic acids from N-substituted N -(4imidazole-ethyl)thiourea by hepatic FMO were also determined. Large differences in Km and Vmax /Km values were found (Table 2), ranging from a Km value of 1011 ± 372 M and a Vmax /Km value of 0.00943 ± 0.00049 ml/(min mg) for 3 to a Km value of 20±11 M and Vmax /Km value of 0.17464±0.07997 ml/(min mg)
R.C.A. Onderwater et al. / Toxicology 197 (2004) 81–91 Table 2 Thiourea-dependent oxidation of thiocholine (n = 3) Compound no.
Km (M)
1 2 3 4 5 6 7 8 9 10 11 12 ␣-Naphthylthiourea
761 664 1011 194 262 176 44 35 20 463 301 29 3.0
± ± ± ± ± ± ± ± ± ± ± ± ±
171 35 372 21 31 57 5 10 11 46 126 4 0.3
Vmax /Km (ml/(min kg)) 0.01342 0.00676 0.00943 0.01595 0.01539 0.01971 0.07880 0.10830 0.17464 0.00675 0.00428 0.09615 0.54
± ± ± ± ± ± ± ± ± ± ± ± ±
0.00072 0.00014 0.00049 0.00110 0.00063 0.00310 0.00662 0.02341 0.07997 0.00024 0.00081 0.00629 0.02
Determined in rat liver microsomes according to Guo and Ziegler (1991).
for 9. Among a homologous series max of N-p-phenylsubstituted N -(4-imidazole-ethyl)thiourea Vmax /Km values could be ranked in the following order: 9 > 8 > 7 > 6 > 5. Km and Vmax /Km values of 12 were found to be similar to those for 8. The Km and Vmax /Km values of N-alkyl- and N-alkylphenyl-substituted N -(4-imidazole-ethyl)thiourea were found to be similar to or even higher than those for the non-cytotoxic compound 5. In Fig. 7, Lineweaver–Burk plots for the formation of the thiol-reactive sulfenic acid metabolite by hepatic FMO enzymes from p-substituted N-phenyl, N (4-imidazole-ethyl)thiourea are shown. As can be seen, all plots intersect at the Y-axis indicating that Vmax values for the p-substituted N-phenyl, N -(4imidazole-ethyl)thiourea are identical, which is expected from the unusual catalytic cycle of the FMO enzyme (Ziegler, 1993). From Tables 1 and 2 and Fig. 5 it can be concluded that a correlation exists between cytotoxicity towards precision-cut rat liver slices and Vmax /Km values for the formation of sulfenic acids from N-substituted N -(4-imidazole-ethyl)thiourea. The compound with the highest Vmax /Km value for the formation of sulfenic acids, 9, was also the most cytotoxic. Compounds with a significantly lower Vmax /Km value for the formation of sulfenic acids, 12, 8, and 7, were less cytotoxic than 9. Compounds with a Vmax /Km value for the formation of sulfenic acids lower than 0.0788 ml/(min mg) were found to be non-cytotoxic
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towards precision-cut rat liver slices for concentrations up to 1000 M and an exposure of 6 h. A similar correlation between cytotoxicity and Vmax /Km value for the formation of sulfenic acids by hepatic FMO was not found for mono- and di-substituted thiourea-containing compounds in freshly prepared rat hepatocytes (Onderwater et al., 1998). However, Km values for hepatic FMO of the thiourea-containing compounds used in this study were much lower. Therefore, differences in stability and the resultant reactivity of the reactive intermediates formed were proposed to be the determinant of STR found. These differences in stability and reactivity might also be a possible explanation for the STR found in the present study. Davis et al. (1985) showed that the high reactivity of benzenesulfenic acids can be reduced by introducing the possibility of intramolecular hydrogen bonding of the sulfenic acid moiety and by reducing the electron density of the sulfenic acid sulfur atom by introducing electron-withdrawing substituents to the benzene ring at the ortho- or para-positions. Decker et al. (1992) suggested that the potential for intramolecular hydrogen bonding to be a determining factor in the stability of the sulfenic acids formed as initial metabolites from a series of thiourea moiety-containing benzimidazoline-2thiones. In conclusion, this study has shown that N-phenyl substituted N -(4-imidazole-ethyl)thiourea-containing electron-withdrawing p-substituents are cytotoxic towards precision-cut rat liver slices. Cytotoxicity is increased with increasing electron withdrawing capacity of the p-substituent. A correlation exists between Vmax /Km value for the formation of sulfenic acids by rat liver FMO enzymes and cytotoxicity.
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