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In vitro neurotoxic assessment of antitumour drugs
Cancer Letter, Elsevier
20 (1983)
Scientific
Ireland
IN VITRO NEUROTOXIC
STEPHEN
A. WHATLEYa
75
75-82
Publishers
Ltd.
ASSESSMENT OF ANTITUMOUR
and BRIDGET
DRUGS
T. HILLb
aDepartment of Neurochemistry, Institute of Neurology, Queen Square, London WClN 3BG and bLaboratory of Cellular Chemotherapy, Imperial Cancer Research Fund, Lincolns Inn Fields, London WCZA 2PX (U.K.) (Received 28 March, 1983) (Revised version received 3 May 1983) (Accepted 26 May 1983)
SUMMARY
We have developed a rapid, specific, in vitro method for quantitatively assessing neurotoxicity of antitumour drugs and related compounds. Using cultures of foetal rat hypothalamic neurones and a staining procedure which specifically identifies neuronal cells, the neurotoxicity of 8 antitumour drugs has been evaluated. The order of neurotoxicity appears to correlate well with their known relative clinical toxicities. Neurotoxicity of the radiosensitizer misonidazole was also identified using this system. This method appears to provide a valuable preclinical screen for neurotoxicity which may be particularly useful in the development of new antitumour drug analogues and radiosensitizers.
INTRODUCTION
Neurological manifestations of toxicity may occur during the use of a wide range of chemotherapeutic agents in cancer treatment [9,12,14,20,21]. These agents may affect both the peripheral nervous system, resulting in symptoms such as reflex suppression, paraesthesiae and gastrointestinal disorders [ 111, and also the central nervous system producing convulsions, lethargy and confusion [ 201. For some drugs, such as 5fluorouracil newotoxicity is uncommon [20], however, for others, such as vincristine, neurotoxicity can present serious limitations on their usage [21]. As new and more potent drugs are developed assessment of their neurotoxic potential will be important in their preclinical evaluation. Animal tests, however, are time-consuming, difficult to quantitate and are influenced by pharmacokinetic variables amongst different species. Therefore the development of simple in vitro screening procedures will be valuable. Such a system using cultured rat midbrain cells has been described ] 111, but this system suffered
0304-3835/83/$03.00 Puhlirhd
and Printd
0 1983 in Trnlanrl
Elsevier
Scientific
Publishers
Ireland
Ltd.
76
from several disadvantages, chiefly that specific neuronal toxicity was not assessed. We have used cultures of foetal rat hypothalamic neurones to assess quantitatively the neurotoxic effects of various antitumour agents and one of the nitroimidazole radiosensitizers. The results obtained suggest that this system may provide a valuable, rapid pre-screening test for neurotoxicity. MATERIALS
AND METHODS
Materials Sources of the drugs used for this study and their methods of solubilisation for in vitro testing are listed in Table 1. Sterile cell culture medium and its supplements were obtained from Gibco Biocult (Renfrewshire, Scotland), except for fluorodeoxyuridine, which was obtained from the Sigma Chemical Company (London). All other chemicals were purchased from BDH (Dorset, U.K.) as ‘Analar’ grade where possible. Preparation of cell cultures Primary cultures of foetal rat hypothalamic neurones were prepared as described previously [22]. Briefly, the hypothalamic area was dissected aseptically from brains of rat embryos of 16 days gestation. After trypsiniza-
TABLE 1 DRUGS USED AND THEIR SOURCES Drug
Source
cis-Platinuma Dibromodulcitol
Gift from Bristol-Myers Co. Ltd., Slough, Bucks, U.K. The Development Therapeutics Program, Chemotherapy, N.C.I.,Bethesda,MD, U.S.A. Roche Products Ltd., Welwyn Garden City, Herts, U.K. Gift from E.R. Squibb & Sons Ltd., Wirral, Merseyside, U.K. Gifts from Eli Lilly & Co. Ltd., Basingstoke, Hants, U.K.
5-Fluorouracil Hydroxyurea Vincristine, vinblastine, vindesine DDMP Misonidazole
Gifts from Wellcome Research Labs., Beckenham, Kent, U.K. Gift from Dr. I. Stratford, Institute of Cancer Research, Sutton, Surrey, U.K.
All the drugs were dissolved in either saline or growth medium without serum with the exception of dibromodulcitol, which was dissolved in 10% dimethyl sulphoxide before dilution to not more than 1% in culture and DDMP, which was dissolved in 2.5% ethanolit HCl. acis-Platinum, cis-dichlorodiamine platinum (II).
77
tion (0.25% trypsin, 13 min at 37°C) the tissue was dissociated by trituration with a Pasteur pipette in 2 stages. Dissociated cells were resuspended in growth medium and plated at a density of 2.2 X 10’ cells/cm’ onto a preexistent confluent ‘feeder’ layer of rat embryonic non-neuronal hypothalamic cells. Cells were cultured in a humidified 10% COz, 90% air atmosphere in Dulbecco’s modified Eagles medium supplemented with non-essential amino acids, 10% heat inactivated foetal calf serum, and penicillin/streptomycin (25 units/ml). In addition, fluorodeoxyuridine was added to the growth medium at 8 X lo-’ M, a concentration which prevented the growth of non-neuronal cells but which was not toxic to neuronal cells [22]. Under these culture conditions, after 1 day in vitro, presumptive neurones were easily recognised by their rounded, highly refractive cell bodies, large nucleus relative to cytoplasm and long processes. These cells were more rigorously identified as neuronal by specific stains. Thus they were heavily stained by thionine, methylene blue and silver impregnation, whereas the underlying cell layer was not [ 221. For neuronal counting, neurones were conveniently identified by heavy thionine staining. When the primary cell suspension was seeded onto polylysine coated coverslips without the presence of a ‘feeder’ layer, over 95% of the attached cells were neuronal in characteristic after 1 day in vitro, indicating a high proportion of neuronal cells in the original cell suspension. Toxicity
measurements
Drugs were added to cell cultures 3 days after plating. After 24 h incubation, the cells were immediately fixed with 10% formalin in phosphate buffered saline. Duplicate coverslips were removed for staining with thionine and, after staining, positive (neuronal) cells were counted by inverting the coverslip over a haemocytometer [22]. A minimum of either 500 cells or 1 cm2 surface area were counted in random fields on each coverslip. Treated cultures are expressed as a percentage of control cell density (nontreated or solvent-treated cultures). The mean f S.E. of cell density for replicate cultures was calculated and survival curves plotted on a logarithmic scale. RESULTS
Dose response curves following a 24-h exposure to various antitumour drugs are shown in Fig. 1. In this experimental system percent control cell density is directly equivalent to cell kill since the neuronal cells are nonmitotic. Untreated cultures show a stable neuronal density over the experimental period [ 221. Most of the drugs tested displayed a dose response curve which consisted of an initial shoulder where the drugs were not detectably toxic, followed at higher concentrations by a region characterised by progressively increasing
Fig. 1. Effects of a 24-h exposure to a range of specific drug concentrations cell density.
on neuronal
cell kill. However, hydroxyurea and 2,4-diamino-5-( 3’,4’-dichlorophenyl)methylpyrimidine (DDMP) could not be shown to be toxic in this system at the restricted concentrations tested. These concentrations were limited by the problems of osmolarity changes with hydroxyurea and insolubility with DDMP. For these drugs, longer times of exposure or the measurement of parameters other than cell killing may be required to detect any neurotoxic potential, Vincristine, vinblastine and vindesine all demonstrated a logarithmic phase of cell kill at higher drug concentrations. For vinblastine, however, a consistently reproducible plateau region was reached by about 90% cell kill, indicating limited cell killing effectiveness for this fixed exposure time. It is notable that the concentration of vincristine needed to produce a given level of cell kill is less than that of vinblastine and vindesine, indicating its greater neurotoxicity. These vinca alkaloids were not detectably toxic to the non-neuronal feeder layer at any of the concentrations used. In contrast, c&platinum and dibromodulcitol showed signs of toxicity to non-neuronal cells at the higher drug concentrations tested, so that it was not possible to quantitate neuronal cell kill in excess of 1 log with these drugs. The effects of the radiosensitizer misonidazole on neuronal cells is shown in Fig. 2. This drug proved toxic at doses above 2 mg/ml. In an attempt to establish whether the sensitivity of normal hypothalamic neurones in culture to these drugs could provide information on their neuro-
79
Fig. 2. Effects of a 24-h exposure to a range of misonidazole concentrations cell density.
on neuronal
toxic potential in vivo, where cytotoxic drug doses are selected for effective tumour cell kill, the concentrations required to reduce survival of hypothalamic neurones were compared with those known to reduce effectively the survival of human neuroblastoma cells in vitro. Table 2 compares the TABLE 2 A COMPARISON OF DRUG CONCENTRATIONS REQUIRED TO REDUCE THE SURVIVAL OF RAT HYPOTHALAMIC NEURONES AND HUMAN NEUROBLASTOMA CELLS BY 70% IN VITRO Drug
Vincristine Vinblastine Vindesine cisPlatinum Dibromodulcitol DDMP Hydroxyurea 5-Fluorouracil
Concentration for 70% cell kill (g/ml) (ID,,) Neurone@
Neuroblastomab
0.0041 0.036 0.048 5.1 140 >2.69 > 3800 2160
0.0016 0.006’ 0.0037 0.145 6.1 0.0042c 16.8 3.9
a Established from the data given in Fig. 1. b Taken from Ref. 8. c Established from new data obtained from Ref. 8.
ID,, neurones/ID,, neuroblastoma
2.6 6.0 12.9 35.2 23.0 > 600 > 225 554
80
concentrations required to reduce the survival of hypothalamic neurones by 70% with those required to produce the same extent of cell kill in human neuroblastoma cells [8]. The ratio of these two dose levels, given in the last column of the table, provides a suggested measure of risk to CNS neurones over the effective range of the drugs for CNS tumours and possibly other tumour type. Using these data, it is possible to segregate the drugs into three classes; those with a low ratio or high ‘risk’ (vincristine, vinblastine and vindesine); those with a medium ratio or medium ‘risk’ (cisplatinum, dibromodulcitol) and those with a high ratio or low ‘risk’ (5-fluorouracil, hydroxyurea, DDMP). DISCUSSION
The toxicity of chemical agents to the nervous system has been assessed using physiological, behavioural and neurochemical criteria as well as by morphological [ 131 and electrophysiological [4,5] techniques, which have also been used to study antitumour agent toxicities. However, all these techniques are complex and time consuming. Cell culture systems of nervous tissue have offered the opportunity for rapid, simple measurements of toxicity which may be confirmed in vivo [11,15]. The culture system used in this report has several advantages over other dissociated cell culture systems. Firstly, both neuronal and non-neuronal cell populations are stable over the experimental period in untreated cultures, thus any reduction in neuronal numbers is directly equivalent to cell kill. In addition, the presence of a stable non-neuronal cell population both minimises effects of antimitotic agents on the glial cells and also reduces effects on neuronal cells due simply to inhibition of glial cell division, since neuronal survival and differentiation is dependent on glial cell presence [ 1,16,22]. Secondly, we have established neuronal cell number by a staining procedure which specifically identifies neuronal cells [ 221. With this system, we have quantitated toxic effects of vincristine, vinblastine, vindesine, cis-platinum, dibromodulcitol and misonidazole. The order of neurotoxicity of the three vinca alkaloids correlates with their relative clinical toxicities, vincristine being the most toxic in vivo [10,12,18]. Therefore, although their relative clinical toxicity has also been attributed to pharmacokinetic differences [ 121, we have confirmed [ 10,111 that there is also a cellular basis for the neurotoxic order of these drugs. By comparing these neurotoxic effects with their effectiveness in growth inhibition of neuroblastoma cells in vitro, we have classified the drugs into three categories depending on their dose ratios: the vinca alkaloids are assessed as having a high risk during effective tumour treatment, cis-platinum and dibromodulcitol as having a medium risk, and 5-fluorouracil, hydroxyurea and DDMP as having a low risk. These classifications correlate with clinical experience, the vinca alkaloids being known to have high neural toxicity which can limit their use [ 211, and &s-platinum [6,14] and dibromo-
dulcitol [ 2 ] having medium toxicity. In contrast, 5-fluorouracil, hydroxyurea and DDMP present neurological problems infrequently [7,20,21]. In addition, we have been able to confirm the in vivo neurotoxicity of the radiosensitizer misonidazole [3,17]. Attempts to relate in vitro drug sensitivity to clinical response are complicated by the lack of adequate human pharmacokinetic data. Assessments of neurotoxic drug levels therefore need to be supplemented by measurement of plasma, cerebrospinal fluid, and brain levels of drugs in order to assess neurotoxic potential. However, the assessment of risk factors at effective doses against tumour cells in vitro may be a means of obtaining more reliable measurements of in vivo risk which could be confirmed later by pharmacological studies. These present assessments are made with the reservation that the use of only one neuroblastoma cell line may not be representative of chemotherapy response in other neuroblastomas. The use of other neuroblastoma cell lines and other tumour cell types would be valuable in assessing neurotoxic potential in the treatment of both brain tumours and, linked with pharmacological studies, other tumour types. However, with these reservations in mind we have been able to confirm clinical experience with these antitumour drugs. This system may therefore represent a valuable rapid pre-screening test for the neurotoxic potential of antitumour agents. This system may also prove valuable in assessing improvements in the development of new vinca alkaloids and radiosensitizers, neurotoxicity being a problem which may be overcome by the development of novel analogues for both of these classes of drug [11,19]. ACKNOWLEDGEMENTS
The authors are pleased to acknowledge the technical expertise of R.D.H. Whelan in carrying out the drug sensitivity assays on the human neuroblastoma cells, the secretarial assistance of Miss Daksha Gandhi and the preparation of the artwork by Mrs Audrey Symons and the I.C.R.F. Photographic Department and all the pharmaceutical companies who provided drug samples for this study. REFERENCES 1 Banker, G.A. and Cowan, W.M. (1977) Rat hippocampal neurons in dispersed cell culture. Brain Res., 126, 397-425. 2 Belej, M.A., Troetel, W.M., Weiss, A.J., Stambaugh, J.E. and Manthei, R.W. (1972) The absorption and metabolism of dibromodulcitol in patients with advanced cancer. Clin. Pharmacol. Ther., 13, 563-572. 3 Donald Chapman, J. (1979) Hypoxic sensitizers - implications for radiation therapy. N. Engl. J. Med., 301,1429-1432. 4 Edwards, MS., Bolger, C.A., Levin, V.A., Phillips, T.L. and Jewett, D.L. (1982) Evaluation of misonidazole peripheral neurotoxicity in rats by analysis of nerve trains evoked response. Int. J. Radiat. Oncol., Biol. Phys., 8, 69-74.
82 5 Fox, D.A., Lowndes, H.E. and Bierkamper, G.G. (1982) Electrophysiological techniques in neurotoxicology. In: Nervous System Toxicology, pp. 299-315. Editor: C.L. Mitchell. Raven Press, New York. 6 Hemphill, M., Pestrouk, A., Walsh, T., Parhad, I., Clark, A. and Rosenshien, N. (1980) Sensory neuropathy in cis-platinum chemotherapy. Neurology, 30, 429. 7 Hill, B.T. and Price, L.A. (1980) DDMP (2,4-diamino-5-( 3’,4’-dichlorophenyl)-methyl pyrimidine). Cancer Treat. Rev., 7, 95-112. 8 Hill, B.T. and Whelan, R.D.H. (1981) Assessments of the sensitivities of cultured human neuroblastoma cells to antitumour drugs. Pediatr. Res., 15, 1117-1122. 9 Hill, B.T., Whatley, S.A., Bellamy, A.S., Jenkins, L.Y. and Whelan, R.D.H. (1982) Cytotoxic effects and biological activity of 2-Aza-8 germanspiro 4,5-decane-2-propanamine8,8-diethyl-N,N-dimethyl dichloride (NSC 192965; spirogermanium) in vitro. Cancer Res., 42, 2852-2856. 10 Iqbal, Z. and Ochs, S. (1980) Uptake of Vinca alkaloids into mammalian nerve and its subcellular components. J. Neurochem., 34,59-68. 11 King, K.L. and Boder, G.B. (1979) Correlation of the clinical neurotoxicity of the Vinca alkaloids vincristine, vinblastine and vindesine with their effects on cultured rat midbrain cells. Cancer Chemother. Pharmacol., 2,239-242. 12 Nelson, R.L., Dyke, R.W. and Root, M.A. (1980) Comparative pharmacokinetics of vindesine, vincristine and vinblastine in patients with cancer. Cancer Treat. Rev., 7 (Suppl.), 17-24. 13 Norton, S. (1982) Behaviour versus morphology as an indicator of central nervous system toxicity. In: Nervous System Toxicology, pp. 247-273. Editor: C.L. Mitchell. Raven Press, New York. 14 Rozencweig, M., Von Hoff, D.D., Abele, R. and Muggia, F.M. (1980) Cis-platin. In: Cancer Chemotherapy, pp. 107-117. Editor: H.M. Pinedo. Excerpta Medica, Amsterdam. 15 Schrier, B.K. (1982) Nervous system cultures as toxicological test systems. In: Nervous System Toxicology, pp. 337-348. Ed. C.L. Mitchell, Raven Press, New York. 16 Sensenbrenner, M. and Mandel, P. (1974) Behaviour of neuroblasts in the presence of glial cells, fibroblasts and meningeal cells in culture. Exp. Cell Res., 87, 159-167. 17 Urtasun, R.C., Chapman, J#., Feldstein, M.L., Band, R.P., Rabin, H.R., Wilson, A.F., Marynowski, B., Starreveld, E. and Shnitka, T. (1978) Peripheral neuropathy related to misonidaxole: incidence and pathology. Br. J. Cancer, 37 (Suppl.), 271-275. 18 Valdivieso, M. (1980) Phase I and II studies of vindisine. Cancer Treat. Rev., 7 (Suppl.), 31-37. 19 Wasserman, T.H. (1981) Hypoxic cell radiosensitizers - present and future. Int. J. Radiat. Oncol., Biol. Phys., 7,849-852. 20 Weiss, H.D., Walker, M.D. and Wiernik, P.H. (1974) Neurotoxicity of commonly used antineoplastic agents. N. Engl. J. Med., 290, 75-81. 21 Weiss, H.D., Walker, M.D. and Wiernik, P.H. (1974) Neurotoxicity of commonly used antineoplastic agents. N. Engl. J. Med., 290, 127-134. 22 Whatley, S.A., Hall, C. and Lim, L. (1981) Hypothalamic neurons in dissociated cell culture: the mechanism of increased survival times in the presence of non-neuronal cells. J. Neurochem., 36,2052-2056.
20 (1983)
Scientific
Ireland
IN VITRO NEUROTOXIC
STEPHEN
A. WHATLEYa
75
75-82
Publishers
Ltd.
ASSESSMENT OF ANTITUMOUR
and BRIDGET
DRUGS
T. HILLb
aDepartment of Neurochemistry, Institute of Neurology, Queen Square, London WClN 3BG and bLaboratory of Cellular Chemotherapy, Imperial Cancer Research Fund, Lincolns Inn Fields, London WCZA 2PX (U.K.) (Received 28 March, 1983) (Revised version received 3 May 1983) (Accepted 26 May 1983)
SUMMARY
We have developed a rapid, specific, in vitro method for quantitatively assessing neurotoxicity of antitumour drugs and related compounds. Using cultures of foetal rat hypothalamic neurones and a staining procedure which specifically identifies neuronal cells, the neurotoxicity of 8 antitumour drugs has been evaluated. The order of neurotoxicity appears to correlate well with their known relative clinical toxicities. Neurotoxicity of the radiosensitizer misonidazole was also identified using this system. This method appears to provide a valuable preclinical screen for neurotoxicity which may be particularly useful in the development of new antitumour drug analogues and radiosensitizers.
INTRODUCTION
Neurological manifestations of toxicity may occur during the use of a wide range of chemotherapeutic agents in cancer treatment [9,12,14,20,21]. These agents may affect both the peripheral nervous system, resulting in symptoms such as reflex suppression, paraesthesiae and gastrointestinal disorders [ 111, and also the central nervous system producing convulsions, lethargy and confusion [ 201. For some drugs, such as 5fluorouracil newotoxicity is uncommon [20], however, for others, such as vincristine, neurotoxicity can present serious limitations on their usage [21]. As new and more potent drugs are developed assessment of their neurotoxic potential will be important in their preclinical evaluation. Animal tests, however, are time-consuming, difficult to quantitate and are influenced by pharmacokinetic variables amongst different species. Therefore the development of simple in vitro screening procedures will be valuable. Such a system using cultured rat midbrain cells has been described ] 111, but this system suffered
0304-3835/83/$03.00 Puhlirhd
and Printd
0 1983 in Trnlanrl
Elsevier
Scientific
Publishers
Ireland
Ltd.
76
from several disadvantages, chiefly that specific neuronal toxicity was not assessed. We have used cultures of foetal rat hypothalamic neurones to assess quantitatively the neurotoxic effects of various antitumour agents and one of the nitroimidazole radiosensitizers. The results obtained suggest that this system may provide a valuable, rapid pre-screening test for neurotoxicity. MATERIALS
AND METHODS
Materials Sources of the drugs used for this study and their methods of solubilisation for in vitro testing are listed in Table 1. Sterile cell culture medium and its supplements were obtained from Gibco Biocult (Renfrewshire, Scotland), except for fluorodeoxyuridine, which was obtained from the Sigma Chemical Company (London). All other chemicals were purchased from BDH (Dorset, U.K.) as ‘Analar’ grade where possible. Preparation of cell cultures Primary cultures of foetal rat hypothalamic neurones were prepared as described previously [22]. Briefly, the hypothalamic area was dissected aseptically from brains of rat embryos of 16 days gestation. After trypsiniza-
TABLE 1 DRUGS USED AND THEIR SOURCES Drug
Source
cis-Platinuma Dibromodulcitol
Gift from Bristol-Myers Co. Ltd., Slough, Bucks, U.K. The Development Therapeutics Program, Chemotherapy, N.C.I.,Bethesda,MD, U.S.A. Roche Products Ltd., Welwyn Garden City, Herts, U.K. Gift from E.R. Squibb & Sons Ltd., Wirral, Merseyside, U.K. Gifts from Eli Lilly & Co. Ltd., Basingstoke, Hants, U.K.
5-Fluorouracil Hydroxyurea Vincristine, vinblastine, vindesine DDMP Misonidazole
Gifts from Wellcome Research Labs., Beckenham, Kent, U.K. Gift from Dr. I. Stratford, Institute of Cancer Research, Sutton, Surrey, U.K.
All the drugs were dissolved in either saline or growth medium without serum with the exception of dibromodulcitol, which was dissolved in 10% dimethyl sulphoxide before dilution to not more than 1% in culture and DDMP, which was dissolved in 2.5% ethanolit HCl. acis-Platinum, cis-dichlorodiamine platinum (II).
77
tion (0.25% trypsin, 13 min at 37°C) the tissue was dissociated by trituration with a Pasteur pipette in 2 stages. Dissociated cells were resuspended in growth medium and plated at a density of 2.2 X 10’ cells/cm’ onto a preexistent confluent ‘feeder’ layer of rat embryonic non-neuronal hypothalamic cells. Cells were cultured in a humidified 10% COz, 90% air atmosphere in Dulbecco’s modified Eagles medium supplemented with non-essential amino acids, 10% heat inactivated foetal calf serum, and penicillin/streptomycin (25 units/ml). In addition, fluorodeoxyuridine was added to the growth medium at 8 X lo-’ M, a concentration which prevented the growth of non-neuronal cells but which was not toxic to neuronal cells [22]. Under these culture conditions, after 1 day in vitro, presumptive neurones were easily recognised by their rounded, highly refractive cell bodies, large nucleus relative to cytoplasm and long processes. These cells were more rigorously identified as neuronal by specific stains. Thus they were heavily stained by thionine, methylene blue and silver impregnation, whereas the underlying cell layer was not [ 221. For neuronal counting, neurones were conveniently identified by heavy thionine staining. When the primary cell suspension was seeded onto polylysine coated coverslips without the presence of a ‘feeder’ layer, over 95% of the attached cells were neuronal in characteristic after 1 day in vitro, indicating a high proportion of neuronal cells in the original cell suspension. Toxicity
measurements
Drugs were added to cell cultures 3 days after plating. After 24 h incubation, the cells were immediately fixed with 10% formalin in phosphate buffered saline. Duplicate coverslips were removed for staining with thionine and, after staining, positive (neuronal) cells were counted by inverting the coverslip over a haemocytometer [22]. A minimum of either 500 cells or 1 cm2 surface area were counted in random fields on each coverslip. Treated cultures are expressed as a percentage of control cell density (nontreated or solvent-treated cultures). The mean f S.E. of cell density for replicate cultures was calculated and survival curves plotted on a logarithmic scale. RESULTS
Dose response curves following a 24-h exposure to various antitumour drugs are shown in Fig. 1. In this experimental system percent control cell density is directly equivalent to cell kill since the neuronal cells are nonmitotic. Untreated cultures show a stable neuronal density over the experimental period [ 221. Most of the drugs tested displayed a dose response curve which consisted of an initial shoulder where the drugs were not detectably toxic, followed at higher concentrations by a region characterised by progressively increasing
Fig. 1. Effects of a 24-h exposure to a range of specific drug concentrations cell density.
on neuronal
cell kill. However, hydroxyurea and 2,4-diamino-5-( 3’,4’-dichlorophenyl)methylpyrimidine (DDMP) could not be shown to be toxic in this system at the restricted concentrations tested. These concentrations were limited by the problems of osmolarity changes with hydroxyurea and insolubility with DDMP. For these drugs, longer times of exposure or the measurement of parameters other than cell killing may be required to detect any neurotoxic potential, Vincristine, vinblastine and vindesine all demonstrated a logarithmic phase of cell kill at higher drug concentrations. For vinblastine, however, a consistently reproducible plateau region was reached by about 90% cell kill, indicating limited cell killing effectiveness for this fixed exposure time. It is notable that the concentration of vincristine needed to produce a given level of cell kill is less than that of vinblastine and vindesine, indicating its greater neurotoxicity. These vinca alkaloids were not detectably toxic to the non-neuronal feeder layer at any of the concentrations used. In contrast, c&platinum and dibromodulcitol showed signs of toxicity to non-neuronal cells at the higher drug concentrations tested, so that it was not possible to quantitate neuronal cell kill in excess of 1 log with these drugs. The effects of the radiosensitizer misonidazole on neuronal cells is shown in Fig. 2. This drug proved toxic at doses above 2 mg/ml. In an attempt to establish whether the sensitivity of normal hypothalamic neurones in culture to these drugs could provide information on their neuro-
79
Fig. 2. Effects of a 24-h exposure to a range of misonidazole concentrations cell density.
on neuronal
toxic potential in vivo, where cytotoxic drug doses are selected for effective tumour cell kill, the concentrations required to reduce survival of hypothalamic neurones were compared with those known to reduce effectively the survival of human neuroblastoma cells in vitro. Table 2 compares the TABLE 2 A COMPARISON OF DRUG CONCENTRATIONS REQUIRED TO REDUCE THE SURVIVAL OF RAT HYPOTHALAMIC NEURONES AND HUMAN NEUROBLASTOMA CELLS BY 70% IN VITRO Drug
Vincristine Vinblastine Vindesine cisPlatinum Dibromodulcitol DDMP Hydroxyurea 5-Fluorouracil
Concentration for 70% cell kill (g/ml) (ID,,) Neurone@
Neuroblastomab
0.0041 0.036 0.048 5.1 140 >2.69 > 3800 2160
0.0016 0.006’ 0.0037 0.145 6.1 0.0042c 16.8 3.9
a Established from the data given in Fig. 1. b Taken from Ref. 8. c Established from new data obtained from Ref. 8.
ID,, neurones/ID,, neuroblastoma
2.6 6.0 12.9 35.2 23.0 > 600 > 225 554
80
concentrations required to reduce the survival of hypothalamic neurones by 70% with those required to produce the same extent of cell kill in human neuroblastoma cells [8]. The ratio of these two dose levels, given in the last column of the table, provides a suggested measure of risk to CNS neurones over the effective range of the drugs for CNS tumours and possibly other tumour type. Using these data, it is possible to segregate the drugs into three classes; those with a low ratio or high ‘risk’ (vincristine, vinblastine and vindesine); those with a medium ratio or medium ‘risk’ (cisplatinum, dibromodulcitol) and those with a high ratio or low ‘risk’ (5-fluorouracil, hydroxyurea, DDMP). DISCUSSION
The toxicity of chemical agents to the nervous system has been assessed using physiological, behavioural and neurochemical criteria as well as by morphological [ 131 and electrophysiological [4,5] techniques, which have also been used to study antitumour agent toxicities. However, all these techniques are complex and time consuming. Cell culture systems of nervous tissue have offered the opportunity for rapid, simple measurements of toxicity which may be confirmed in vivo [11,15]. The culture system used in this report has several advantages over other dissociated cell culture systems. Firstly, both neuronal and non-neuronal cell populations are stable over the experimental period in untreated cultures, thus any reduction in neuronal numbers is directly equivalent to cell kill. In addition, the presence of a stable non-neuronal cell population both minimises effects of antimitotic agents on the glial cells and also reduces effects on neuronal cells due simply to inhibition of glial cell division, since neuronal survival and differentiation is dependent on glial cell presence [ 1,16,22]. Secondly, we have established neuronal cell number by a staining procedure which specifically identifies neuronal cells [ 221. With this system, we have quantitated toxic effects of vincristine, vinblastine, vindesine, cis-platinum, dibromodulcitol and misonidazole. The order of neurotoxicity of the three vinca alkaloids correlates with their relative clinical toxicities, vincristine being the most toxic in vivo [10,12,18]. Therefore, although their relative clinical toxicity has also been attributed to pharmacokinetic differences [ 121, we have confirmed [ 10,111 that there is also a cellular basis for the neurotoxic order of these drugs. By comparing these neurotoxic effects with their effectiveness in growth inhibition of neuroblastoma cells in vitro, we have classified the drugs into three categories depending on their dose ratios: the vinca alkaloids are assessed as having a high risk during effective tumour treatment, cis-platinum and dibromodulcitol as having a medium risk, and 5-fluorouracil, hydroxyurea and DDMP as having a low risk. These classifications correlate with clinical experience, the vinca alkaloids being known to have high neural toxicity which can limit their use [ 211, and &s-platinum [6,14] and dibromo-
dulcitol [ 2 ] having medium toxicity. In contrast, 5-fluorouracil, hydroxyurea and DDMP present neurological problems infrequently [7,20,21]. In addition, we have been able to confirm the in vivo neurotoxicity of the radiosensitizer misonidazole [3,17]. Attempts to relate in vitro drug sensitivity to clinical response are complicated by the lack of adequate human pharmacokinetic data. Assessments of neurotoxic drug levels therefore need to be supplemented by measurement of plasma, cerebrospinal fluid, and brain levels of drugs in order to assess neurotoxic potential. However, the assessment of risk factors at effective doses against tumour cells in vitro may be a means of obtaining more reliable measurements of in vivo risk which could be confirmed later by pharmacological studies. These present assessments are made with the reservation that the use of only one neuroblastoma cell line may not be representative of chemotherapy response in other neuroblastomas. The use of other neuroblastoma cell lines and other tumour cell types would be valuable in assessing neurotoxic potential in the treatment of both brain tumours and, linked with pharmacological studies, other tumour types. However, with these reservations in mind we have been able to confirm clinical experience with these antitumour drugs. This system may therefore represent a valuable rapid pre-screening test for the neurotoxic potential of antitumour agents. This system may also prove valuable in assessing improvements in the development of new vinca alkaloids and radiosensitizers, neurotoxicity being a problem which may be overcome by the development of novel analogues for both of these classes of drug [11,19]. ACKNOWLEDGEMENTS
The authors are pleased to acknowledge the technical expertise of R.D.H. Whelan in carrying out the drug sensitivity assays on the human neuroblastoma cells, the secretarial assistance of Miss Daksha Gandhi and the preparation of the artwork by Mrs Audrey Symons and the I.C.R.F. Photographic Department and all the pharmaceutical companies who provided drug samples for this study. REFERENCES 1 Banker, G.A. and Cowan, W.M. (1977) Rat hippocampal neurons in dispersed cell culture. Brain Res., 126, 397-425. 2 Belej, M.A., Troetel, W.M., Weiss, A.J., Stambaugh, J.E. and Manthei, R.W. (1972) The absorption and metabolism of dibromodulcitol in patients with advanced cancer. Clin. Pharmacol. Ther., 13, 563-572. 3 Donald Chapman, J. (1979) Hypoxic sensitizers - implications for radiation therapy. N. Engl. J. Med., 301,1429-1432. 4 Edwards, MS., Bolger, C.A., Levin, V.A., Phillips, T.L. and Jewett, D.L. (1982) Evaluation of misonidazole peripheral neurotoxicity in rats by analysis of nerve trains evoked response. Int. J. Radiat. Oncol., Biol. Phys., 8, 69-74.
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