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Immunotoxicity of ethyl-4-isothiocyanatobutanoate in male Wistar rats
Toxicology 145 (2000) 217 – 225
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Immunotoxicity of ethyl-4-isothiocyanatobutanoate in male Wistar rats J. Tulinska a,*, A. Sovcikova b, A. Liskova a, J. Kubova a, K. Horakova b a
Department of Immunotoxicology, Institute of Pre6enti6e and Clinical Medicine, Limbo6a 14, 833 01 Bratisla6a, Slo6ak Republic b Faculty of Chemical Technology, Slo6ak Technological Uni6ersity, Radlinskeho 9, Bratisla6a, Slo6ak Republic Received 30 July 1999; accepted 11 December 1999
Abstract The immunotoxicity of ethyl-4-isothiocyanatobutanoate (E-4IB) using different immuno-pathological parameters and immune function assays in male Wistar rats was evaluated. The rats were administered intraperitoneally 12 times with E-4IB in three varying doses of 21, 28 and 35 mg/kg of body weight, over a period of 36 days. The doses of E-4IB were set according to the results of previous experiments by its anti-proliferative activity in vivo. High and medium doses of E-4IB exceeded the maximum tolerated dose after the 36-day treatment period. Symptoms of toxicity were displayed by a drop in body weight, spleen and thymus weight and in organ and bone marrow cellularity. Haematological changes displayed a dose-dependent decrease in the percentage of lymphocytes and dose-dependent increase in the percentage of polymorphonuclear leukocytes in peripheral blood. The white blood cell count in rats exposed to a high dose of E-4IB was suppressed. The immune system of rats administered 21 mg/kg of E-4IB (low dose) was unaffected. No changes in primary antibody response to sheep erythrocytes, in vitro proliferative response of spleen lymphocytes to mitogens and phagocytic activity of leukocytes were found in those rats. Our findings indicate that this newly developed anti-cancer drug is not immunotoxic. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Immunotoxicity; Ethyl-4-isothiocyanatobutanoate (E-4IB); Rat; Isothiocyanates (ITC); Cytotoxic effect; Cancerostatic effect
1. Introduction Immunotoxicology, a rapidly developing subdiscipline of toxicology, involves the study of
* Corresponding author. Fax: +421-7-54773906. E-mail address: [email protected] (J. Tulinska)
interactions of xenobiotics with the immune system, drugs included. The objective of this study was to ascertain the immune parameters of rats exposed to ethyl-4-isothiocyanatobutanoate (E4IB). Isothiocyanates (ITCs) are known as natural or synthetic anti-fungal (Drobnica et al., 1967), anti-bacterial (Ono et al., 1998), cytotoxic (Hora´kova´ et al., 1989; Nastruzzi et al., 1996) and anti-tumour agents (Hora´kova´ et al., 1993; Pintao
0300-483X/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 9 9 ) 0 0 2 2 5 - 5
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et al., 1995; Hecht 1996) and their anthelmintic activity both in humans and animals (Leland et al., 1967) was described. Some ITCs are commonly found in human nutrition, e.g. allyl ITC and phenyl ITC (Musk et al., 1995). In healthy intact plants, ITCs are present in undetectable volumes, but their progenitors are present, and together with a molecule of glucose and sulphate they form heteroglycosides called glucosinolates (GL), especially in families of dicotyledonous angiosperms and with all species of the large family Cruciferae. The GL represent one of the most extensive and widely studied groups of sulphur organic compounds of plant origin (Drobnica et al., 1977). Knowledge of their structure, biosynthesis and degradation, analytical determination and relations to systematic botany have been presented in review of Ettlinger and Kjaer (1968). The structure of ITCs, as well as their binding activity represented by the isothiocyanate group (NCS), is important with respect to their biological properties. Variances in the cytotoxicity of certain kinds of natural and synthetic ITCs were observed (Hora´kova´ et al., 1968a,b). Esters of isothiocyanatocarboxylic acids are among the most active compounds of the group (Hora´kova´ et al., 1971, 1989). The most pronounced cytotoxicity on HeLa and V79 cells was found with 4-methylisothiocyanatobutanoate, 5ethylisothiocyanatopentanoate (Somorovska´ et al., 1994) and ethyl-4-isothiocyanatobutanoate (E4IB), a derivative of gama-aminobutyric acid (GABA), which is synthesised from hydrochloride GABA ester with thiophosgene in the chloroform:water system (Floch and Kova´cˇ 1975). Remarkable cytotoxic activity in vitro suggests possible cancerostatic potential of E-4IB. Since it did not display any anti-microbial activity, this compound is anticipated to have specific cytotoxic properties. An Ames test proved that E-4IB showed no mutagenic effect (Hora´kova´ et al., 1989). E-4IB had an inhibitory effect upon division of HeLa cells in the concentration range of 0.1 – 1.0 mg/l, and simultaneously enhanced cell proliferation and increased DNA accumulation in cells (Hora´kova´ et al., 1993). It can thus be assumed
that the inhibition on the division of HeLa cells is particularly due to the effect of E-4IB on the cell cycle and that E-4IB only secondarily affects the energetic processes in the cell. Moreover, E-4IB displays anti-proliferative activity in vivo on transplanted sarcoma B77-RF cells in rats (Hora´kova´ et al., 1993). These results support our hypothesis that E-4IB has a potential anti-tumour effect. In view of this, the aims of our further experiments were: 1. to study the effect of E-4IB on cellular and humoral immunity in rats, 2. to suggest a new therapeutic scheme for E-4IB in treatment of experimentally induced fibrosarcoma in rats, taking into account results from immune assays in animals exposed to E-4IB, 3. to review the use of immunotoxicological screening assays for the testing of xenobiotics for pre-clinical drug research.
2. Material and methods
2.1. Experimental animals Outbred male SPF Wistar rats (Velaz, Prague), body weight 210–250 g were used. After 1 week of acclimatisation the animals were divided into four groups, 20 animals each. Rats, housed five per polycarbonate cage, were fed a standard laboratory diet (DOS-2b, Velaz, Prague) and had water ad libitum. Ambient temperature of 229 2°C, relative humidity of 509 5% and 12 h light/dark cycle were maintained.
2.2. Dosage E-4IB dissolved in 80% dimethylsulphoxide (DMSO) was administered intraperitoneally for 36 days in 12 doses. During the first 2 weeks E-4IB was administered every second day, altogether six times. In the next 3 weeks the substance was dosed every fourth day, altogether six times. Groups of rats (20 animals per group) were treated with E-4IB in three different doses: 21 mg/kg/day (low dose), 28 mg/kg/day (medium dose) and 35 mg/kg/day (high dose), correspond-
J. Tulinska et al. / Toxicology 145 (2000) 217–225
ing to 15, 20 and 25% of the LD50 value, respectively. Control animal group (20 animals) was dosed with 80% DMSO.
2.3. Haematology and organ cellularity Erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume and leukocyte count were determined using haematological analyser (Coulter counter). Differential leukocyte count was assessed from blood smears stained according to Giemsa-Romanowski by counting 200 leukocytes. Spleen, thymus and bone marrow cellularities were determined by counting using microscope.
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DNA synthesis, 1 mCi of 3H-thymidine was added to each well 24 h before the end of the incubation period. Thereafter, cultures were harvested by suction on glass-fibre filters (Dynatech), using a multiple automated sample harvester (Dynatech). Radioactivity was measured in a liquid scintillation spectrometer and expressed as disintegrations/min/culture.
2.6. Phagocytic acti6ity assay Phagocytic activity was assayed by the method of Fornu˚sek et al. (1981) based on uptake of synthetic methacrylate hydrophilic particles by polymorphonuclear leukocytes and monocytes (UVVVR Prague).
2.4. Plaque forming cell (PFC) assay 2.7. Statistical analysis Separate groups of rats were immunised with a single intravenous injection of 1 ml of sheep red blood cells (SRBC) 2 ×108/ml in sterile saline. Four days later the number of plaque forming cells (PFC) was determined (Cunningham and Szenberg, 1968). The splenocytes were diluted with E-MEM culture medium (Sigma) to give a final concentration 1:100. The guinea pig complement was diluted with culture medium to a 10% solution and SRBC to a 4% solution. Cell suspensions were incubated in a humidified atmosphere for 1 h at 37°C.
2.5. Lymphoproliferation assay Spleen suspensions were prepared by pressing the spleen between the frozen microscope slides under sterile conditions. Counts of cells were adjusted to 2×106/ml with RPMI 1640 medium (Sigma), supplemented with 10% foetal calf serum, 50 mg/ml gentamicin and 2 mM L-glutamine. One hundred microlitres of diluted cell suspension were dispensed into 96-well flat bottom culture plates. Two mitogens: Concanavalin A (ConA), (Sigma) in final concentrations of 1, 5, 10 mg/ml and mitogen from Salmonella typhimurium (STM), (Sigma) in concentrations of 10, 50 and 100 mg/ml were used. Cultures were set up in triplicates and incubated for 72 h at 37°C in humid atmosphere of 5% CO2 in air. To assess
Differences between control and exposed groups were evaluated using one-way analysis of variance (ANOVA).
3. Results
3.1. General obser6ations All animals survived the experimental period of 36 days and any E-4IB-related deaths were observed. Transient weakness (10 min) was recorded after administration of the high dose of E-4IB in 25% of the animals. The rats displayed normal gait and fur texture, and there was no drainage from any body orifice.
3.2. Body and organ weights Table 1 shows the mean body weight of the animals in individual groups on the 1st, 8th, 15th, 22nd, 29th and 36th day of the experiment. In the third week of study, the body weight of highly dosed animals was 8% lower compared to control rats. In the fifth week of the experiment, this difference was even more pronounced (12%). Suppression of body weight was statistically significant at the end of the experiment in rats treated with medium and high doses of E-4IB.
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A 5-week period of administrating E-4IB resulted in changes in lymphoid organ weight (Table 2). Rats dosed with medium and high doses of E-4IB had reduced absolute spleen and thymus weights. Similar changes were recorded in relative thymus weight. Relative spleen weight
was depressed only in animals treated with medium doses of E-4IB. The absolute popliteal lymph node weight decreased in rats given medium doses of E-4IB. No changes in relative weight of this lymphoid organ were observed.
Table 1 Body weight in male outbred Wistar rats intraperitoneally exposed to twelve doses of ethyl-4-isothiocyanatobutanoate (E-4IB) for 36 daysa Day Day 1 Day 8 Day 15 Day 22 Day 29 Day 36
a
Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE
Vehicle
E-4IB: 21 mg/kg
E-4IB: 28 mg/kg
E-4IB: 35 mg/kg
231.00 5.52 253.00 6.76 276.00 7.33 296.00 7.99 315.00 9.55 331.50 10.08
230.00 5.40 247.22 5.41 268.33 5.83 286.67 6.61 297.78 6.51 310.00 8.94
230.50 7.24 251.50 8.13 268.00 7.90 283.00 8.79 288.50 10.49 296.00* 11.22
229.50 4.44 239.50 5.35 256.50 5.87 272.00 6.29 281.50 7.34 291.67* 8.90
SE-standard error, statistically significant differences are marked with asterisks: * PB0.05.
Table 2 Absolute and relative organ weights in male Wistar rats intraperitoneally exposed to twelve doses of ethyl-4-isothiocyanatobutanoate (E-4IB) for 36 daysa Parameter Spleen (g) % of body weight Thymus (g) % of body weight Liver (g) % of body weight Popliteal lymph nodes (g) % of body weight Mesenterial lymph nodes (g) % of body weight
a
Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE
Vehicle
E-4IB: 21 mg/kg
E-4IB: 28 mg/kg
E-4IB: 35 mg/kg
0.696 0.036 0.204 0.009 0.630 0.027 0.185 0.008 10.61 0.54 3.11 1.64 0.00527 0.00047 0.00154 0.00013 0.01290 0.00114 0.00386 0.00030
0.690 0.028 0.225 0.011 0.578 0.038 0.190 0.010 9.78 0.39 3.26 0.09 0.00560 0.00019 0.00186 0.00006 0.01251 0.00112 0.00387 0.00027
0.504*** 0.023 0.171** 0.008 0.382*** 0.030 0.129*** 0.010 9.83 0.28 3.28 0.11 0.00409** 0.00017 0.00153 0.00022 0.01165 0.00070 0.00397 0.00026
0.535*** 0.031 0.190 0.011 0.460*** 0.040 0.156*** 0.011 9.34 0.24 3.29 0.11 0.00448 0.00033 0.00152 0.00007 0.01010 0.00050 0.00360 0.00023
SE-standard error, statistically significant differences are marked with asterisks: ** PB0.01, *** PB0.001.
J. Tulinska et al. / Toxicology 145 (2000) 217–225
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Table 3 Haematologic parameters and organ cellularity in male Wistar rats intraperitoneally exposed to twelve doses of ethyl-4-isothiocyanatobutanoate (E-4IB) for 36 daysa Parameter Erythrocytes (×1012/l) Hemoglobin (g/dl) Hematocrit (%) Mean cell volume (fl) Leukocytes (×109/l) % Lymphocytes % Polymorphonucear leukocytes % Monocytes % Large granular lymphocytes Bone marrow cellularity (×106) Thymus cellularity (×106)
a
Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE
Vehicle
E-4IB: 21 mg/kg
E-4IB: 28 mg/kg
E-4IB: 35 mg/kg
5.74 0.62 12.10 1.00 50.0 5.0 87.20 1.87 15.16 4.35 74.11 1.55 14.22 1.42 1.70 0.42 8.60 0.34 240.0 32.0 155.0 45.0
5.63 0.56 11.80 0.80 49.0 7.0 87.10 2.02 12.03 2.79 67.22 3.70 18.80 3.60 2.30 0.33 7.80 0.59 237.0 37.0 132.0 30.0
5.45 0.59 11.70 1.30 48.0 5.0 89.00 5.23 12.07 5.15 60.00*** 2.45 28.22*** 2.80 2.70 0.40 8.20 0.94 233.0 58.0 93.0** 38.0
5.77 0.82 11.30 1.50 51.0 8.0 88.22 3.42 10.64* 3.07 55.63*** 2.30 33.00*** 2.08 2.56 0.56 7.44 0.97 199.0* 43.0 91.0** 51.0
SE-standard error, statistically significant differences are marked with asterisks: *PB0.05, **PB0.01,***PB0.001.
A slight dose-dependent decrease of absolute mesenterial lymph node weight was recorded in dosed rats, contrary to an unchanged relative mesenterial lymph node weight.
3.3. Haematology and organ cellularity A decrease in the leukocyte count was found only in animals exposed to the high doses of E-4IB (Table 3). Animals exposed to the high and medium doses of E-4IB displayed a dose-dependent decrease in the percentage of lymphocytes (Table 3). In the percentage of polymorphonuclear leukocytes, changes were contrary. There were no changes in the percentage of large granular lymphocytes. The percentage of monocytes slightly rose in all dosed groups. No changes were observed in the number and mean cell volume of erythrocytes, haemoglobin level and haematocrit in treated rats. A pronounced suppressive effect of E-4IB was observed in the thymus and spleen cellularity in
animals treated with medium and high E-4IB doses (Table 3). The bone marrow cellularity decreased in rats given high dose of E-4IB.
3.4. Primary antibody response to sheep erythrocytes A slight increase in plaque-forming cells (PFC) per million spleen cells in rats treated with low doses of E-4IB was observed (Fig. 1). In animals exposed to high dose, a significant decrease in the number of PFC per spleen was recorded.
3.5. Proliferati6e acti6ity of T- and B-lymphocytes in the spleen A decrease in proliferative activity of spleen T-lymphocytes was observed in rats exposed to the high dose of E-4IB (Fig. 2). Suppression was statistically significant only in cultures stimulated by 1 mg/ml of Concanavalin A.
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The values of proliferative activity of Blymphocytes are shown in Fig. 3. Exposure to all doses of E-4IB slightly and non-significantly decreased the proliferative activity of B-lymphocytes in splenocyte cultures.
3.6. Phagocytic acti6ity of leukocytes No changes in phagocytic activity were found, either in monocytes or in polymorphonuclear leukocytes (Fig. 4).
4. Discussion In this study, the immunotoxic potential of E-4IB in Wistar rats was evaluated. The dosages of E-4IB were set according to the results of previous experiments (Hora´kova´ et al., 1993) by its anti-proliferative activity in vivo. As the administration of E-4IB in a dose of 14 mg/kg of body weight did not reduce the tumour weight, the lowest dose in our study was set at 21 mg/kg/ bw (15% LD50). The medium dose, 28 mg/kg/bw
Fig. 1. Primary antibody response to T-dependent antigen in male Wistar rats intraperitoneally exposed to twelve doses of ethyl-4-isothiocyanatobutanoate (E-4IB) for 36 days. Bars indicate standard errors. SC (E6)-spleen cellularity expressed in millions, PFC/milion SC-number of plaque forming cells per million spleen cells, PFC (E3)/spleen-number of plaque forming cells in thousands per all spleen cells. Statistically significant differences are marked with asterisks: *P B0.05.
Fig. 2. Proliferative capacity of T-lymphocytes in male Wistar rats intraperitoneally exposed to twelve doses of ethyl-4-isothiocyanatobutanoate (E-4IB) for 36 days. Bars indicate standard errors. Spleen lymphocytes were stimulated with mitogen Concanavalin A (Con A) and incorporation of [3H]-thymidine into replicating cells was measured. Results are expressed as disintegrations per min/culture. Statistically significant differences are marked with asterisks: *PB 0.05.
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Fig. 3. Proliferative capacity of B-lymphocytes in male Wistar rats intraperitoneally exposed to twelve doses of ethyl-4-isothiocyanatobutanoate (E-4IB) for 36 days. Bars indicate standard errors. Spleen lymphocytes were stimulated with mitogen from Salmonella typhimurium (STM) and incorporation of [3H]-thymidine into replicating cells was measured. Results are expressed as disintegrations per min/culture.
Fig. 4. Phagocytic activity of polymorphonuclear leukocytes and monocytes in peripheral blood in male Wistar rats intraperitoneally exposed to twelve doses of ethyl-4-isothiocyanatobutanoate (E-4IB) for 36 days. Bars indicate standard errors. Data are expressed as percentage of phagocytic polymorphonuclear leukocytes (PMN/PMN) and monocytes (MONO/MONO). PMN + MONO is common percentage of phagocytic cells.
(20% of LD50), of E-4IB in our experiment corresponds to the high dose used in previous experiments. Hora´kova´ et al. (1993) administered E-4IB five times. In order to prolong the treatment period, we administered E-4IB twelve times. To attain a higher drug level in the initial phase of the treatment we applied E-4IB every second day during the first two weeks, altogether six times. In the following three weeks, the application interval was prolonged i.e. every fourth day, altogether six times. Approaches to testing the immunotoxicity of xenobiotics in animals are heterogenous and dis-
cussed (Vos 1980; Luster et al., 1988; Macela et al., 1989; Van Loveren and Vos 1989; Descotes 1992). When choosing the panel of assays to assess the immunotoxic potential of E-4IB in Wistar rats, we took into consideration the criteria set out by the International Programme on Chemical Safety (ICICIS Report, 1998). The basic parameter of toxicity-decreased body weight and suppression of spleen and thymus weight was observed during the fifth week of administration of medium and high doses of E-4IB. Organ cellularity matched the toxic effect better. Results indicated that the reduction in organ weight was accompanied by a lower cell count.
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Importance of bone marrow cellularity, being a source of precursors of immunocompetent cells, was shown in the study of De Waal et al. (1995) who used Cyclosporin A as a model chemical to follow its effect on the bone marrow. In this context, it is important that the cellularity of bone marrow was decreased in the animal group treated with high doses of E-4IB. E-4IB treatment resulted in an expressive response of peripheral compartment of the immune system in blood, e.g. a significant reduction in the leukocyte count in the highly dosed group and dose-dependent increase of polymorphonuclear leukocytes and dose-dependent decrease of lymphocytes. Nevertheless, no evidence of changes was found either in the phagocytic activity of polymorphonuclear leukocytes or monocytes in exposed animals. These results show that phagocytosis, as the oldest immune protecting mechanism, seems to be very resistent to the damage caused by xenobiotics. The observed slight increase in B-lymphocyte activity in the group treated with low dose of E-4IB can be considered as an intensified defence of the organism when exposed to a low dose of the xenobiotic. As a result of marked cellularity reduction in spleen found in groups administered the toxic medium and high doses of E-4IB, a similar reduction in the number of plaque-forming cells (PFC) in the spleen could be observed. With regard to the PFC assay method, such data show that in order to obtain more valid information on primary antibody response of spleen cells, the plaque production should be expressed both per standardised cell number (PFC/million spleen cells) and per spleen cellularity (PFC/spleen). In conclusion, the medium and high doses of E-4IB given 12 times to male Wistar rats, exceeded the maximum tolerated dose after a 36-day treatment period. Symptoms of toxicity were displayed by a reduction in body weight, spleen and thymus weight and cellularity. The immune system of rats dosed 21 mg/kg of E-4IB (low dose) was not affected. Our findings indicate that this newly developed anti-cancer drug is not immunotoxic. It can also be concluded that the used panel of immunotoxicity tests, based on oral administra-
tion of the tested xenobiotic and as recommended by the OECD, can also be implemented in preclinical research of newly developed drugs administered intraperitoneally. Acknowledgements We would like to express our gratitude to Helena Turazova´ and Viera Vacha´lkova´ for their excellent technical help. This work was supported by the Grant Agency of Slovak Republic, grant c 04.92.37. References Cunningham, A.J., Szenberg, A., 1968. Further improvements in the plaque technique for detecting single antibody-forming cells. Immunology 14, 559 – 600. Descotes, J., 1992. Immunotoxicology of immunomodulators. Dev. Biol. Stand. 77, 99 – 102. De Waal, E.J., Timmerman, H.H., Dortant, P.M., Kranjc, M.A., Van Loveren, H., 1995. Investigation of a screening battery for immunotoxicity of pharmaceuticals within a 28-day oral toxicity study using azathioprine and cyclosporin A model compounds. Regul. Toxicol. Pharmacol. 3, 327 – 338. Drobnica, L., Zemanova´, M., Nemec, P., Antosˇ, K., Kristia´n, P., Stullerova´, A., Knoppova´, V., 1967. Antifungal activity of isothiocyanates and related compounds. I. Naturally occuring isothiocyanates and their related analogues. Appl. Microbiol. 15, 701. Drobnica, L., Kristia´n, P., Augustı´n, J., 1977. The chemistry of cyanates and their thioderivates. In: The Chemistry of the-NCS Group. Part 2, pp. 1003 – 1199. Ettlinger, M.G., Kjaer, A., 1968. In: Mabry, T.J., Alston, R.E., Runeckles, V.C. (Eds.), Recent advances in phytochemistry. Vol.1, North-Holland Publ. Co., Amsterdam, Appleton-Century-Crofts, Diu. Meredith Corp., New York, pp. 59 – 144. Floch, L., Kova´cˇ, S., 1975. Synthesis and properties of alkylisothiocyanatocarboxylates. Coll. Czech. Chem. Commun. 40, 2845. Fornu˚sek, L., Veˇtvicˇka, V., Kopecˇek, E., 1981. Differences in phagocytic activity of methacrylate copolymer particles in normal and stimulated macrophages. Experientia 37, 418 – 420. Hecht, S.S., 1996. Chemoprevention of lung cancer by isothiocyanates. Adv. Exp. Med. Biol. 401, 1 – 11. Hora´kova´, K., Drobnica, L., Nemec, P., Antosˇ, K., Kristia´n, P., 1968a. Cytotoxic and cancerostatic activity of isothiocyanates and related compounds. I. Activity of some naturally occuring isothiocyanates and their synthetic analogues on HeLa cells. Neoplasma 15, 169 – 176.
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Immunotoxicity of ethyl-4-isothiocyanatobutanoate in male Wistar rats J. Tulinska a,*, A. Sovcikova b, A. Liskova a, J. Kubova a, K. Horakova b a
Department of Immunotoxicology, Institute of Pre6enti6e and Clinical Medicine, Limbo6a 14, 833 01 Bratisla6a, Slo6ak Republic b Faculty of Chemical Technology, Slo6ak Technological Uni6ersity, Radlinskeho 9, Bratisla6a, Slo6ak Republic Received 30 July 1999; accepted 11 December 1999
Abstract The immunotoxicity of ethyl-4-isothiocyanatobutanoate (E-4IB) using different immuno-pathological parameters and immune function assays in male Wistar rats was evaluated. The rats were administered intraperitoneally 12 times with E-4IB in three varying doses of 21, 28 and 35 mg/kg of body weight, over a period of 36 days. The doses of E-4IB were set according to the results of previous experiments by its anti-proliferative activity in vivo. High and medium doses of E-4IB exceeded the maximum tolerated dose after the 36-day treatment period. Symptoms of toxicity were displayed by a drop in body weight, spleen and thymus weight and in organ and bone marrow cellularity. Haematological changes displayed a dose-dependent decrease in the percentage of lymphocytes and dose-dependent increase in the percentage of polymorphonuclear leukocytes in peripheral blood. The white blood cell count in rats exposed to a high dose of E-4IB was suppressed. The immune system of rats administered 21 mg/kg of E-4IB (low dose) was unaffected. No changes in primary antibody response to sheep erythrocytes, in vitro proliferative response of spleen lymphocytes to mitogens and phagocytic activity of leukocytes were found in those rats. Our findings indicate that this newly developed anti-cancer drug is not immunotoxic. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Immunotoxicity; Ethyl-4-isothiocyanatobutanoate (E-4IB); Rat; Isothiocyanates (ITC); Cytotoxic effect; Cancerostatic effect
1. Introduction Immunotoxicology, a rapidly developing subdiscipline of toxicology, involves the study of
* Corresponding author. Fax: +421-7-54773906. E-mail address: [email protected] (J. Tulinska)
interactions of xenobiotics with the immune system, drugs included. The objective of this study was to ascertain the immune parameters of rats exposed to ethyl-4-isothiocyanatobutanoate (E4IB). Isothiocyanates (ITCs) are known as natural or synthetic anti-fungal (Drobnica et al., 1967), anti-bacterial (Ono et al., 1998), cytotoxic (Hora´kova´ et al., 1989; Nastruzzi et al., 1996) and anti-tumour agents (Hora´kova´ et al., 1993; Pintao
0300-483X/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 9 9 ) 0 0 2 2 5 - 5
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et al., 1995; Hecht 1996) and their anthelmintic activity both in humans and animals (Leland et al., 1967) was described. Some ITCs are commonly found in human nutrition, e.g. allyl ITC and phenyl ITC (Musk et al., 1995). In healthy intact plants, ITCs are present in undetectable volumes, but their progenitors are present, and together with a molecule of glucose and sulphate they form heteroglycosides called glucosinolates (GL), especially in families of dicotyledonous angiosperms and with all species of the large family Cruciferae. The GL represent one of the most extensive and widely studied groups of sulphur organic compounds of plant origin (Drobnica et al., 1977). Knowledge of their structure, biosynthesis and degradation, analytical determination and relations to systematic botany have been presented in review of Ettlinger and Kjaer (1968). The structure of ITCs, as well as their binding activity represented by the isothiocyanate group (NCS), is important with respect to their biological properties. Variances in the cytotoxicity of certain kinds of natural and synthetic ITCs were observed (Hora´kova´ et al., 1968a,b). Esters of isothiocyanatocarboxylic acids are among the most active compounds of the group (Hora´kova´ et al., 1971, 1989). The most pronounced cytotoxicity on HeLa and V79 cells was found with 4-methylisothiocyanatobutanoate, 5ethylisothiocyanatopentanoate (Somorovska´ et al., 1994) and ethyl-4-isothiocyanatobutanoate (E4IB), a derivative of gama-aminobutyric acid (GABA), which is synthesised from hydrochloride GABA ester with thiophosgene in the chloroform:water system (Floch and Kova´cˇ 1975). Remarkable cytotoxic activity in vitro suggests possible cancerostatic potential of E-4IB. Since it did not display any anti-microbial activity, this compound is anticipated to have specific cytotoxic properties. An Ames test proved that E-4IB showed no mutagenic effect (Hora´kova´ et al., 1989). E-4IB had an inhibitory effect upon division of HeLa cells in the concentration range of 0.1 – 1.0 mg/l, and simultaneously enhanced cell proliferation and increased DNA accumulation in cells (Hora´kova´ et al., 1993). It can thus be assumed
that the inhibition on the division of HeLa cells is particularly due to the effect of E-4IB on the cell cycle and that E-4IB only secondarily affects the energetic processes in the cell. Moreover, E-4IB displays anti-proliferative activity in vivo on transplanted sarcoma B77-RF cells in rats (Hora´kova´ et al., 1993). These results support our hypothesis that E-4IB has a potential anti-tumour effect. In view of this, the aims of our further experiments were: 1. to study the effect of E-4IB on cellular and humoral immunity in rats, 2. to suggest a new therapeutic scheme for E-4IB in treatment of experimentally induced fibrosarcoma in rats, taking into account results from immune assays in animals exposed to E-4IB, 3. to review the use of immunotoxicological screening assays for the testing of xenobiotics for pre-clinical drug research.
2. Material and methods
2.1. Experimental animals Outbred male SPF Wistar rats (Velaz, Prague), body weight 210–250 g were used. After 1 week of acclimatisation the animals were divided into four groups, 20 animals each. Rats, housed five per polycarbonate cage, were fed a standard laboratory diet (DOS-2b, Velaz, Prague) and had water ad libitum. Ambient temperature of 229 2°C, relative humidity of 509 5% and 12 h light/dark cycle were maintained.
2.2. Dosage E-4IB dissolved in 80% dimethylsulphoxide (DMSO) was administered intraperitoneally for 36 days in 12 doses. During the first 2 weeks E-4IB was administered every second day, altogether six times. In the next 3 weeks the substance was dosed every fourth day, altogether six times. Groups of rats (20 animals per group) were treated with E-4IB in three different doses: 21 mg/kg/day (low dose), 28 mg/kg/day (medium dose) and 35 mg/kg/day (high dose), correspond-
J. Tulinska et al. / Toxicology 145 (2000) 217–225
ing to 15, 20 and 25% of the LD50 value, respectively. Control animal group (20 animals) was dosed with 80% DMSO.
2.3. Haematology and organ cellularity Erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume and leukocyte count were determined using haematological analyser (Coulter counter). Differential leukocyte count was assessed from blood smears stained according to Giemsa-Romanowski by counting 200 leukocytes. Spleen, thymus and bone marrow cellularities were determined by counting using microscope.
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DNA synthesis, 1 mCi of 3H-thymidine was added to each well 24 h before the end of the incubation period. Thereafter, cultures were harvested by suction on glass-fibre filters (Dynatech), using a multiple automated sample harvester (Dynatech). Radioactivity was measured in a liquid scintillation spectrometer and expressed as disintegrations/min/culture.
2.6. Phagocytic acti6ity assay Phagocytic activity was assayed by the method of Fornu˚sek et al. (1981) based on uptake of synthetic methacrylate hydrophilic particles by polymorphonuclear leukocytes and monocytes (UVVVR Prague).
2.4. Plaque forming cell (PFC) assay 2.7. Statistical analysis Separate groups of rats were immunised with a single intravenous injection of 1 ml of sheep red blood cells (SRBC) 2 ×108/ml in sterile saline. Four days later the number of plaque forming cells (PFC) was determined (Cunningham and Szenberg, 1968). The splenocytes were diluted with E-MEM culture medium (Sigma) to give a final concentration 1:100. The guinea pig complement was diluted with culture medium to a 10% solution and SRBC to a 4% solution. Cell suspensions were incubated in a humidified atmosphere for 1 h at 37°C.
2.5. Lymphoproliferation assay Spleen suspensions were prepared by pressing the spleen between the frozen microscope slides under sterile conditions. Counts of cells were adjusted to 2×106/ml with RPMI 1640 medium (Sigma), supplemented with 10% foetal calf serum, 50 mg/ml gentamicin and 2 mM L-glutamine. One hundred microlitres of diluted cell suspension were dispensed into 96-well flat bottom culture plates. Two mitogens: Concanavalin A (ConA), (Sigma) in final concentrations of 1, 5, 10 mg/ml and mitogen from Salmonella typhimurium (STM), (Sigma) in concentrations of 10, 50 and 100 mg/ml were used. Cultures were set up in triplicates and incubated for 72 h at 37°C in humid atmosphere of 5% CO2 in air. To assess
Differences between control and exposed groups were evaluated using one-way analysis of variance (ANOVA).
3. Results
3.1. General obser6ations All animals survived the experimental period of 36 days and any E-4IB-related deaths were observed. Transient weakness (10 min) was recorded after administration of the high dose of E-4IB in 25% of the animals. The rats displayed normal gait and fur texture, and there was no drainage from any body orifice.
3.2. Body and organ weights Table 1 shows the mean body weight of the animals in individual groups on the 1st, 8th, 15th, 22nd, 29th and 36th day of the experiment. In the third week of study, the body weight of highly dosed animals was 8% lower compared to control rats. In the fifth week of the experiment, this difference was even more pronounced (12%). Suppression of body weight was statistically significant at the end of the experiment in rats treated with medium and high doses of E-4IB.
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A 5-week period of administrating E-4IB resulted in changes in lymphoid organ weight (Table 2). Rats dosed with medium and high doses of E-4IB had reduced absolute spleen and thymus weights. Similar changes were recorded in relative thymus weight. Relative spleen weight
was depressed only in animals treated with medium doses of E-4IB. The absolute popliteal lymph node weight decreased in rats given medium doses of E-4IB. No changes in relative weight of this lymphoid organ were observed.
Table 1 Body weight in male outbred Wistar rats intraperitoneally exposed to twelve doses of ethyl-4-isothiocyanatobutanoate (E-4IB) for 36 daysa Day Day 1 Day 8 Day 15 Day 22 Day 29 Day 36
a
Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE
Vehicle
E-4IB: 21 mg/kg
E-4IB: 28 mg/kg
E-4IB: 35 mg/kg
231.00 5.52 253.00 6.76 276.00 7.33 296.00 7.99 315.00 9.55 331.50 10.08
230.00 5.40 247.22 5.41 268.33 5.83 286.67 6.61 297.78 6.51 310.00 8.94
230.50 7.24 251.50 8.13 268.00 7.90 283.00 8.79 288.50 10.49 296.00* 11.22
229.50 4.44 239.50 5.35 256.50 5.87 272.00 6.29 281.50 7.34 291.67* 8.90
SE-standard error, statistically significant differences are marked with asterisks: * PB0.05.
Table 2 Absolute and relative organ weights in male Wistar rats intraperitoneally exposed to twelve doses of ethyl-4-isothiocyanatobutanoate (E-4IB) for 36 daysa Parameter Spleen (g) % of body weight Thymus (g) % of body weight Liver (g) % of body weight Popliteal lymph nodes (g) % of body weight Mesenterial lymph nodes (g) % of body weight
a
Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE
Vehicle
E-4IB: 21 mg/kg
E-4IB: 28 mg/kg
E-4IB: 35 mg/kg
0.696 0.036 0.204 0.009 0.630 0.027 0.185 0.008 10.61 0.54 3.11 1.64 0.00527 0.00047 0.00154 0.00013 0.01290 0.00114 0.00386 0.00030
0.690 0.028 0.225 0.011 0.578 0.038 0.190 0.010 9.78 0.39 3.26 0.09 0.00560 0.00019 0.00186 0.00006 0.01251 0.00112 0.00387 0.00027
0.504*** 0.023 0.171** 0.008 0.382*** 0.030 0.129*** 0.010 9.83 0.28 3.28 0.11 0.00409** 0.00017 0.00153 0.00022 0.01165 0.00070 0.00397 0.00026
0.535*** 0.031 0.190 0.011 0.460*** 0.040 0.156*** 0.011 9.34 0.24 3.29 0.11 0.00448 0.00033 0.00152 0.00007 0.01010 0.00050 0.00360 0.00023
SE-standard error, statistically significant differences are marked with asterisks: ** PB0.01, *** PB0.001.
J. Tulinska et al. / Toxicology 145 (2000) 217–225
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Table 3 Haematologic parameters and organ cellularity in male Wistar rats intraperitoneally exposed to twelve doses of ethyl-4-isothiocyanatobutanoate (E-4IB) for 36 daysa Parameter Erythrocytes (×1012/l) Hemoglobin (g/dl) Hematocrit (%) Mean cell volume (fl) Leukocytes (×109/l) % Lymphocytes % Polymorphonucear leukocytes % Monocytes % Large granular lymphocytes Bone marrow cellularity (×106) Thymus cellularity (×106)
a
Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE
Vehicle
E-4IB: 21 mg/kg
E-4IB: 28 mg/kg
E-4IB: 35 mg/kg
5.74 0.62 12.10 1.00 50.0 5.0 87.20 1.87 15.16 4.35 74.11 1.55 14.22 1.42 1.70 0.42 8.60 0.34 240.0 32.0 155.0 45.0
5.63 0.56 11.80 0.80 49.0 7.0 87.10 2.02 12.03 2.79 67.22 3.70 18.80 3.60 2.30 0.33 7.80 0.59 237.0 37.0 132.0 30.0
5.45 0.59 11.70 1.30 48.0 5.0 89.00 5.23 12.07 5.15 60.00*** 2.45 28.22*** 2.80 2.70 0.40 8.20 0.94 233.0 58.0 93.0** 38.0
5.77 0.82 11.30 1.50 51.0 8.0 88.22 3.42 10.64* 3.07 55.63*** 2.30 33.00*** 2.08 2.56 0.56 7.44 0.97 199.0* 43.0 91.0** 51.0
SE-standard error, statistically significant differences are marked with asterisks: *PB0.05, **PB0.01,***PB0.001.
A slight dose-dependent decrease of absolute mesenterial lymph node weight was recorded in dosed rats, contrary to an unchanged relative mesenterial lymph node weight.
3.3. Haematology and organ cellularity A decrease in the leukocyte count was found only in animals exposed to the high doses of E-4IB (Table 3). Animals exposed to the high and medium doses of E-4IB displayed a dose-dependent decrease in the percentage of lymphocytes (Table 3). In the percentage of polymorphonuclear leukocytes, changes were contrary. There were no changes in the percentage of large granular lymphocytes. The percentage of monocytes slightly rose in all dosed groups. No changes were observed in the number and mean cell volume of erythrocytes, haemoglobin level and haematocrit in treated rats. A pronounced suppressive effect of E-4IB was observed in the thymus and spleen cellularity in
animals treated with medium and high E-4IB doses (Table 3). The bone marrow cellularity decreased in rats given high dose of E-4IB.
3.4. Primary antibody response to sheep erythrocytes A slight increase in plaque-forming cells (PFC) per million spleen cells in rats treated with low doses of E-4IB was observed (Fig. 1). In animals exposed to high dose, a significant decrease in the number of PFC per spleen was recorded.
3.5. Proliferati6e acti6ity of T- and B-lymphocytes in the spleen A decrease in proliferative activity of spleen T-lymphocytes was observed in rats exposed to the high dose of E-4IB (Fig. 2). Suppression was statistically significant only in cultures stimulated by 1 mg/ml of Concanavalin A.
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The values of proliferative activity of Blymphocytes are shown in Fig. 3. Exposure to all doses of E-4IB slightly and non-significantly decreased the proliferative activity of B-lymphocytes in splenocyte cultures.
3.6. Phagocytic acti6ity of leukocytes No changes in phagocytic activity were found, either in monocytes or in polymorphonuclear leukocytes (Fig. 4).
4. Discussion In this study, the immunotoxic potential of E-4IB in Wistar rats was evaluated. The dosages of E-4IB were set according to the results of previous experiments (Hora´kova´ et al., 1993) by its anti-proliferative activity in vivo. As the administration of E-4IB in a dose of 14 mg/kg of body weight did not reduce the tumour weight, the lowest dose in our study was set at 21 mg/kg/ bw (15% LD50). The medium dose, 28 mg/kg/bw
Fig. 1. Primary antibody response to T-dependent antigen in male Wistar rats intraperitoneally exposed to twelve doses of ethyl-4-isothiocyanatobutanoate (E-4IB) for 36 days. Bars indicate standard errors. SC (E6)-spleen cellularity expressed in millions, PFC/milion SC-number of plaque forming cells per million spleen cells, PFC (E3)/spleen-number of plaque forming cells in thousands per all spleen cells. Statistically significant differences are marked with asterisks: *P B0.05.
Fig. 2. Proliferative capacity of T-lymphocytes in male Wistar rats intraperitoneally exposed to twelve doses of ethyl-4-isothiocyanatobutanoate (E-4IB) for 36 days. Bars indicate standard errors. Spleen lymphocytes were stimulated with mitogen Concanavalin A (Con A) and incorporation of [3H]-thymidine into replicating cells was measured. Results are expressed as disintegrations per min/culture. Statistically significant differences are marked with asterisks: *PB 0.05.
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Fig. 3. Proliferative capacity of B-lymphocytes in male Wistar rats intraperitoneally exposed to twelve doses of ethyl-4-isothiocyanatobutanoate (E-4IB) for 36 days. Bars indicate standard errors. Spleen lymphocytes were stimulated with mitogen from Salmonella typhimurium (STM) and incorporation of [3H]-thymidine into replicating cells was measured. Results are expressed as disintegrations per min/culture.
Fig. 4. Phagocytic activity of polymorphonuclear leukocytes and monocytes in peripheral blood in male Wistar rats intraperitoneally exposed to twelve doses of ethyl-4-isothiocyanatobutanoate (E-4IB) for 36 days. Bars indicate standard errors. Data are expressed as percentage of phagocytic polymorphonuclear leukocytes (PMN/PMN) and monocytes (MONO/MONO). PMN + MONO is common percentage of phagocytic cells.
(20% of LD50), of E-4IB in our experiment corresponds to the high dose used in previous experiments. Hora´kova´ et al. (1993) administered E-4IB five times. In order to prolong the treatment period, we administered E-4IB twelve times. To attain a higher drug level in the initial phase of the treatment we applied E-4IB every second day during the first two weeks, altogether six times. In the following three weeks, the application interval was prolonged i.e. every fourth day, altogether six times. Approaches to testing the immunotoxicity of xenobiotics in animals are heterogenous and dis-
cussed (Vos 1980; Luster et al., 1988; Macela et al., 1989; Van Loveren and Vos 1989; Descotes 1992). When choosing the panel of assays to assess the immunotoxic potential of E-4IB in Wistar rats, we took into consideration the criteria set out by the International Programme on Chemical Safety (ICICIS Report, 1998). The basic parameter of toxicity-decreased body weight and suppression of spleen and thymus weight was observed during the fifth week of administration of medium and high doses of E-4IB. Organ cellularity matched the toxic effect better. Results indicated that the reduction in organ weight was accompanied by a lower cell count.
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Importance of bone marrow cellularity, being a source of precursors of immunocompetent cells, was shown in the study of De Waal et al. (1995) who used Cyclosporin A as a model chemical to follow its effect on the bone marrow. In this context, it is important that the cellularity of bone marrow was decreased in the animal group treated with high doses of E-4IB. E-4IB treatment resulted in an expressive response of peripheral compartment of the immune system in blood, e.g. a significant reduction in the leukocyte count in the highly dosed group and dose-dependent increase of polymorphonuclear leukocytes and dose-dependent decrease of lymphocytes. Nevertheless, no evidence of changes was found either in the phagocytic activity of polymorphonuclear leukocytes or monocytes in exposed animals. These results show that phagocytosis, as the oldest immune protecting mechanism, seems to be very resistent to the damage caused by xenobiotics. The observed slight increase in B-lymphocyte activity in the group treated with low dose of E-4IB can be considered as an intensified defence of the organism when exposed to a low dose of the xenobiotic. As a result of marked cellularity reduction in spleen found in groups administered the toxic medium and high doses of E-4IB, a similar reduction in the number of plaque-forming cells (PFC) in the spleen could be observed. With regard to the PFC assay method, such data show that in order to obtain more valid information on primary antibody response of spleen cells, the plaque production should be expressed both per standardised cell number (PFC/million spleen cells) and per spleen cellularity (PFC/spleen). In conclusion, the medium and high doses of E-4IB given 12 times to male Wistar rats, exceeded the maximum tolerated dose after a 36-day treatment period. Symptoms of toxicity were displayed by a reduction in body weight, spleen and thymus weight and cellularity. The immune system of rats dosed 21 mg/kg of E-4IB (low dose) was not affected. Our findings indicate that this newly developed anti-cancer drug is not immunotoxic. It can also be concluded that the used panel of immunotoxicity tests, based on oral administra-
tion of the tested xenobiotic and as recommended by the OECD, can also be implemented in preclinical research of newly developed drugs administered intraperitoneally. Acknowledgements We would like to express our gratitude to Helena Turazova´ and Viera Vacha´lkova´ for their excellent technical help. This work was supported by the Grant Agency of Slovak Republic, grant c 04.92.37. References Cunningham, A.J., Szenberg, A., 1968. Further improvements in the plaque technique for detecting single antibody-forming cells. Immunology 14, 559 – 600. Descotes, J., 1992. Immunotoxicology of immunomodulators. Dev. Biol. Stand. 77, 99 – 102. De Waal, E.J., Timmerman, H.H., Dortant, P.M., Kranjc, M.A., Van Loveren, H., 1995. Investigation of a screening battery for immunotoxicity of pharmaceuticals within a 28-day oral toxicity study using azathioprine and cyclosporin A model compounds. Regul. Toxicol. Pharmacol. 3, 327 – 338. Drobnica, L., Zemanova´, M., Nemec, P., Antosˇ, K., Kristia´n, P., Stullerova´, A., Knoppova´, V., 1967. Antifungal activity of isothiocyanates and related compounds. I. Naturally occuring isothiocyanates and their related analogues. Appl. Microbiol. 15, 701. Drobnica, L., Kristia´n, P., Augustı´n, J., 1977. The chemistry of cyanates and their thioderivates. In: The Chemistry of the-NCS Group. Part 2, pp. 1003 – 1199. Ettlinger, M.G., Kjaer, A., 1968. In: Mabry, T.J., Alston, R.E., Runeckles, V.C. (Eds.), Recent advances in phytochemistry. Vol.1, North-Holland Publ. Co., Amsterdam, Appleton-Century-Crofts, Diu. Meredith Corp., New York, pp. 59 – 144. Floch, L., Kova´cˇ, S., 1975. Synthesis and properties of alkylisothiocyanatocarboxylates. Coll. Czech. Chem. Commun. 40, 2845. Fornu˚sek, L., Veˇtvicˇka, V., Kopecˇek, E., 1981. Differences in phagocytic activity of methacrylate copolymer particles in normal and stimulated macrophages. Experientia 37, 418 – 420. Hecht, S.S., 1996. Chemoprevention of lung cancer by isothiocyanates. Adv. Exp. Med. Biol. 401, 1 – 11. Hora´kova´, K., Drobnica, L., Nemec, P., Antosˇ, K., Kristia´n, P., 1968a. Cytotoxic and cancerostatic activity of isothiocyanates and related compounds. I. Activity of some naturally occuring isothiocyanates and their synthetic analogues on HeLa cells. Neoplasma 15, 169 – 176.
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