Arch Toxicol (! 992) 66: 224-227
Archives of
Tordcolog 9 Springer-Verlag 1992
Short communication
Cytotoxicity of mebendazole against established cell lines from the human, rat, and mouse liver Futoshi Higa, Keizo Kitsukawa, Michihiro Gaja, Masao Tateyama, Koushin Shikiya, Yoshiteru Shigeno, Fukunori Kinjo, and Atsushi Saito First Department of Internal Medicine, Faculty of Medicine, University of the Ryukyus, 207 Ueham, Nishihara, Okinawa 903-01, Japan Received 4 February 1991/Receivedafter revision 26 September 1991/Accepted7 October 1991
Abstract. The direct cytotoxicity of mebendazole (MBZ) was investigated by using cell lines derived from human, mouse and rat liver. It was demonstrated that Chang liver cells (derived from human liver) were more sensitive to the cytotoxic effects of MBZ than the other two cell lines. Longer incubation of the cells with MBZ resulted in stronger toxicity, and the cytotoxicity was dependent on the MBZ concentration above a certain threshold value (0.25-0.50 rag/1 in a 42-h culture). Inhibition of the proliferation of Chang liver cells by MBZ was detected at a concentration of 0.008 mg/1, a lower concentration than that having a cytotoxic effect. The other two cell lines were less sensitive to the inhibitory effect of MBZ. Proliferation of human mononuclear cells following stimulation by phytohemagglutinin (PHA) was inhibited by MBZ, and this inhibition was more extensive than that of cells stimulated with whole formalin-treated Pseudomonas aeruginosa. It is suggested that dividing ceils may be more sensitive to MBZ cytotoxicity. This anti-proliferative effect may be related to its clinically known side effects, such as hepatotoxicity and bone marrow suppression.
Key words: Mebendazole - Cytotoxicity - Cell lines - In vitro
Introduction Mebendazole (MBZ) is a derivative of benzimidazole, which has a broad-spectrum anthelmintic effect. This drug affects glucose uptake into nematodes (Van den Bossche 1972), and has the ability to bind to tubulin and to inhibit the assembly of microtubules (Van den Bossche 1982). The differential affinity of host and parasite tubulin to MBZ could be one of the factors responsible for its selective toxicity.
Offprint requests to: Futoshi Higa
The toxicity of MBZ had already been evaluated in animal models (rats and dogs) and liver damage was not observed even with long-term administration (Marsb00~ 1973). Its clinical safety has already been reported on elsewhere (Musgrave et al. 1979; Horstmann et al. 1982; Mravak et al. 1983; Luder et al. 1986; Hoegaerden et al 1987). Because of the known safety of the drug and the effectiveness against Strongyloides stercoralis (Grove 1982, 1989) we have been treating the carriers of S. ster. coral& with MBZ at a dose of 200 mg daily for 28 consecutive days. However, unexpected liver damage occurred in about 70% of the treated carriers (Shikiya et al. 1990) Bekhti and Pirotte (1987) also described hepatotoxicity as a side effect of MBZ and its relationship to the serum concentration of the drug, when they gave MBZ (4.5 g/day) a patient suffering from multiple hydatid cysts. The discrepancies between the various reports about the hepatotoxicity of MBZ may result from differences in its pharmacokinetics, its metabolism, and sensitivity to toxicity between the various populations treated. In clinical trials of MBZ performed in patients suffering from echin0coccosis, Luder et al. (1986) described inter-individual variations in the serum level of this drug, and stated that the main reason was variability in absorption. In contrast, hepatic microsomal function and cholestasis have also been reported to be determinants of the plasma MBZ level (Witassek and Bircher 1983). In these experiments, we tried to clarify the relationship between cytotoxicity and the MBZ concentration. We examined how MBZ affected cultured cell lines established from human, mouse, and rat liver. Fresh human hepat0cytes were not used because of their limited availability. Furthermore, the effect of MBZ on the proliferation of human peripheral mononuclear cells was also examined.
Materials and methods Cell lines. The Chang liver cell line (ATCC No.CCL- 13, human), NCTC clone 1469 (ATCC No.CCL-9.1, routine), and BRL-3A (ATCC No.CRL-1442, rat) were purchased from Dai Nippon Pharmaceuticals
225 (Osaka, Japan). They were subcultured and then used for the experiments. The respective culture media for Chang liver, NCTC, and BRL-3A cells were MEM-E, NCTC 135, and F-12K (all from Flow Laboratories Inc. Irvine, Scotland). The media were supplemented with 20 mM N-s-hydroxyethyldiamine-N-2-ethanesulfonic acid (HEPES; Dojin Chemical Laboratories, Ltd, Kumarnoto, Japan), 100 mg/l kanamycin sulfate (Wako Pure Chemicals, Osaka, Japan), and 10% fetal bovineserum (Hazleton, Denver, PA).
Drug. A dose of 200 mg MBZ (provided by Janssen Kyowa Co. Ltd, Tokyo, Japan) was dissolved in 1.0 rnl formic acid, and then diluted with the media for use in the experiments. The formic acid concentration used in these experiments was below 0.005% v/v, at which level the cultured cells were not damaged by this additive (data not shown).
Cytotoxicity testing. Cells were cultured at 106/ml in the presence of 3.7 MBq sodium S]chromate (New England Nuclear, Boston, MA). After 30 min at 37 ~C, the cells were washed three times and re-cultured in quadruplicate in a 96-well fiat bottomed plate (Becton Dickinson, LincolnPark, N J). Culture was performed at a concentration of 1 • 105/well in a final volume of 0.2 ml, to which serial dilutions of MBZ were added. At the indicated times, 0.1 ml of supernatants was removed and 51Cr release was determined with a gamma scintillation counter (Aloka ARC-300, Aloka Co. Ltd, Japan). Maximal release was determined by lysing the cells with saponin (Wako). Spontaneous release was the amount of radioactivity present in the medium of the untreated cells. Cytotoxicity was calculated by the following formula: Cytotoxicity (%) =
(experimental release - spontaneous release) maximum release - spontaneous release
?
~15o x E
~o 8r 9"D -~
6"
INCUBATION TIME
{hour}
Fig. 1. Effects of mebendazole on the proliferation of Chang liver cells. Cells were cultured at 1 • 104/well with 3H-thymidine and the following concentrations of mebendazole (mg/1); 0.0016 ([]), 0.008 (A), 0.04 ( 9 0.2 (B), and 1.0 ( 0 ) . The cells were harvested at the indicated times, and the incorporated 3H-thymidine was counted with a liquid scintillation counter. These data represent the mean + S D of quadruplicate experiments. The asterisks represent a significant difference (p
Archives of
Tordcolog 9 Springer-Verlag 1992
Short communication
Cytotoxicity of mebendazole against established cell lines from the human, rat, and mouse liver Futoshi Higa, Keizo Kitsukawa, Michihiro Gaja, Masao Tateyama, Koushin Shikiya, Yoshiteru Shigeno, Fukunori Kinjo, and Atsushi Saito First Department of Internal Medicine, Faculty of Medicine, University of the Ryukyus, 207 Ueham, Nishihara, Okinawa 903-01, Japan Received 4 February 1991/Receivedafter revision 26 September 1991/Accepted7 October 1991
Abstract. The direct cytotoxicity of mebendazole (MBZ) was investigated by using cell lines derived from human, mouse and rat liver. It was demonstrated that Chang liver cells (derived from human liver) were more sensitive to the cytotoxic effects of MBZ than the other two cell lines. Longer incubation of the cells with MBZ resulted in stronger toxicity, and the cytotoxicity was dependent on the MBZ concentration above a certain threshold value (0.25-0.50 rag/1 in a 42-h culture). Inhibition of the proliferation of Chang liver cells by MBZ was detected at a concentration of 0.008 mg/1, a lower concentration than that having a cytotoxic effect. The other two cell lines were less sensitive to the inhibitory effect of MBZ. Proliferation of human mononuclear cells following stimulation by phytohemagglutinin (PHA) was inhibited by MBZ, and this inhibition was more extensive than that of cells stimulated with whole formalin-treated Pseudomonas aeruginosa. It is suggested that dividing ceils may be more sensitive to MBZ cytotoxicity. This anti-proliferative effect may be related to its clinically known side effects, such as hepatotoxicity and bone marrow suppression.
Key words: Mebendazole - Cytotoxicity - Cell lines - In vitro
Introduction Mebendazole (MBZ) is a derivative of benzimidazole, which has a broad-spectrum anthelmintic effect. This drug affects glucose uptake into nematodes (Van den Bossche 1972), and has the ability to bind to tubulin and to inhibit the assembly of microtubules (Van den Bossche 1982). The differential affinity of host and parasite tubulin to MBZ could be one of the factors responsible for its selective toxicity.
Offprint requests to: Futoshi Higa
The toxicity of MBZ had already been evaluated in animal models (rats and dogs) and liver damage was not observed even with long-term administration (Marsb00~ 1973). Its clinical safety has already been reported on elsewhere (Musgrave et al. 1979; Horstmann et al. 1982; Mravak et al. 1983; Luder et al. 1986; Hoegaerden et al 1987). Because of the known safety of the drug and the effectiveness against Strongyloides stercoralis (Grove 1982, 1989) we have been treating the carriers of S. ster. coral& with MBZ at a dose of 200 mg daily for 28 consecutive days. However, unexpected liver damage occurred in about 70% of the treated carriers (Shikiya et al. 1990) Bekhti and Pirotte (1987) also described hepatotoxicity as a side effect of MBZ and its relationship to the serum concentration of the drug, when they gave MBZ (4.5 g/day) a patient suffering from multiple hydatid cysts. The discrepancies between the various reports about the hepatotoxicity of MBZ may result from differences in its pharmacokinetics, its metabolism, and sensitivity to toxicity between the various populations treated. In clinical trials of MBZ performed in patients suffering from echin0coccosis, Luder et al. (1986) described inter-individual variations in the serum level of this drug, and stated that the main reason was variability in absorption. In contrast, hepatic microsomal function and cholestasis have also been reported to be determinants of the plasma MBZ level (Witassek and Bircher 1983). In these experiments, we tried to clarify the relationship between cytotoxicity and the MBZ concentration. We examined how MBZ affected cultured cell lines established from human, mouse, and rat liver. Fresh human hepat0cytes were not used because of their limited availability. Furthermore, the effect of MBZ on the proliferation of human peripheral mononuclear cells was also examined.
Materials and methods Cell lines. The Chang liver cell line (ATCC No.CCL- 13, human), NCTC clone 1469 (ATCC No.CCL-9.1, routine), and BRL-3A (ATCC No.CRL-1442, rat) were purchased from Dai Nippon Pharmaceuticals
225 (Osaka, Japan). They were subcultured and then used for the experiments. The respective culture media for Chang liver, NCTC, and BRL-3A cells were MEM-E, NCTC 135, and F-12K (all from Flow Laboratories Inc. Irvine, Scotland). The media were supplemented with 20 mM N-s-hydroxyethyldiamine-N-2-ethanesulfonic acid (HEPES; Dojin Chemical Laboratories, Ltd, Kumarnoto, Japan), 100 mg/l kanamycin sulfate (Wako Pure Chemicals, Osaka, Japan), and 10% fetal bovineserum (Hazleton, Denver, PA).
Drug. A dose of 200 mg MBZ (provided by Janssen Kyowa Co. Ltd, Tokyo, Japan) was dissolved in 1.0 rnl formic acid, and then diluted with the media for use in the experiments. The formic acid concentration used in these experiments was below 0.005% v/v, at which level the cultured cells were not damaged by this additive (data not shown).
Cytotoxicity testing. Cells were cultured at 106/ml in the presence of 3.7 MBq sodium S]chromate (New England Nuclear, Boston, MA). After 30 min at 37 ~C, the cells were washed three times and re-cultured in quadruplicate in a 96-well fiat bottomed plate (Becton Dickinson, LincolnPark, N J). Culture was performed at a concentration of 1 • 105/well in a final volume of 0.2 ml, to which serial dilutions of MBZ were added. At the indicated times, 0.1 ml of supernatants was removed and 51Cr release was determined with a gamma scintillation counter (Aloka ARC-300, Aloka Co. Ltd, Japan). Maximal release was determined by lysing the cells with saponin (Wako). Spontaneous release was the amount of radioactivity present in the medium of the untreated cells. Cytotoxicity was calculated by the following formula: Cytotoxicity (%) =
(experimental release - spontaneous release) maximum release - spontaneous release
?
~15o x E
~o 8r 9"D -~
6"
INCUBATION TIME
{hour}
Fig. 1. Effects of mebendazole on the proliferation of Chang liver cells. Cells were cultured at 1 • 104/well with 3H-thymidine and the following concentrations of mebendazole (mg/1); 0.0016 ([]), 0.008 (A), 0.04 ( 9 0.2 (B), and 1.0 ( 0 ) . The cells were harvested at the indicated times, and the incorporated 3H-thymidine was counted with a liquid scintillation counter. These data represent the mean + S D of quadruplicate experiments. The asterisks represent a significant difference (p
