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531. From cell to whole animal
METHODS FOR ASSESSING TOXICITY
291
The 'Skinner box' provides a method of studying the effects of drugs and toxic agents on the behaviour of pigeons. They are trained to perform a task, namely to peck at a key, in order to obtain food. Quantitative criteria of behaviour are provided by the indices of performance, such as number of pecks and frequency of pecking. In fact, with trained birds one obtains records over many months characterized by remarkable precision and reproducibility. A very suitable test material to superimpose on such a background is mercury (Hg) vapour, chronic inhalation of which can lead to the development of tremors, altered behaviour and psychic disturbances in man. Each trained pigeon was exposed in a chamber to an average concentration of 17 mg Hg/m 3 for 2 hr daily, 5 days a week either until it failed to respond or else showed a markedly lower rate of response for 1-3 days; or until it died. When exposure to Hg ceased, the bird was considered to have recovered when the overall response rate proved constant for several weeks. All birds manifested a decrease in response rate, but the time of onset of this effect varied from one to another. In one bird the rate dropped to under 50 % of normal after I week's exposure, rose and fell again in week 4. In 4 pigeons the fall to below 50% took place in week 2 while in 2 others 5 and 6 weeks of exposure were needed. In one hardy warrior the level remained above 50% for 12 weeks. Yet recovery after cessation of exposure bore no relation to the duration of Hg inhalation. The first gross sign of Hg poisoning was tremor, appearing at 5 weeks and becoming more evident during preening, walking and feeding. Weight loss was a late development in birds whose response rates had fallen significantly earlier in the experiment. Analysis of the behavioural effects revealed an early central nervous stimulant action in some birds, but depression was the most prominent general effect. The parallel with human toxic effects of Hg extended to a similar failure to react to environmental stimuli. Although the Maximal Acceptable Concentration for human exposure is 0-1 mg Hg/m 3, exposure of the birds to 17 mg Hg/m 3 failed to produce histological evidence of tissue damage by Hg in the pigeons that died. 531. From cell to whole animal
Smith, C. G., Grady, J. E. & Northam, J. I. (1963). Relationship between cytotoxicity in vitro and whole animal toxicity. Cancer chemother. Rep. No. 30, 9. When a chemical is administered to an animal a very large number of extraneous factors tend to influence the observed biological effect, so that the final outcome may be no reflection of the 'innate' toxicity of the material on living cells. The engaging idea presents itself: why not apply the test material directly to living cells, for example the tissue culture ? This approach assumes even greater importance when we recall that human cell lines, such as liver, can now be maintained successfully in vitro. The obvious difficulty is that cells grown in this way are delicate creatures at the best of times and to wipe them out is not nearly as difficult as to maintain them successfully. Any attempt to correlate cytotoxicity, i.e. inhibition of cell growth or IDs0, with acute LDs0 is therefore of great interest. The authors went further. Since they were dealing with compounds screened for possible use as cancer chemotherapeutic agents they were also able to compare laboratory findings with the dose that proved clinically useful in man. The outcome of the exercise was a statistically significant correlation between inhibition of growth of mammalian cells in tissue culture and acute intraperitoneal toxicity to mice, as well as a correlation with the maximum tolerated dose for man. For any individual compound, however, cytotoxicity was not a useful guide to its behaviour in mice and man. In fact there were some startling discrepancies--for example, fluoroacetate and a barbiturate were innocuous at high levels to cells in vitro; the former has an acute LD50 in mice of 10
292
BIOCHEMICAL PHARMACOLOGY
mg/kg and an estimated oral LDs0 in man of 15 mg/kg. Equally interesting were the IDs0 and LDs0 values (given in that order) for the following pairs of compounds: dimethylformamide >5000, 650; dimethylacetamide >5000, 3000; similarly fumaric acid >1000, 200; 2,4-dich'.orophenoxyacetie acid 45, 838.
BIOCHEMICAL PHARMACOLOGY 532. Mode of action of ethionine Oehlert, W., Kramseh, D. & Beck, V. (1963). Der Einfluss des Abbaues cytoplasmatischer Strukturen auf die DNS-Neubildung bei der experimentellen Athioninvergiftung. Naturwissenschaften 50, 307. Poisoning by ethionine (I), the ethyl homologue of methionine, is known to produce loss of cytoplasmic nucleotides in cells with a high protein metabolism; this also results in decorttposition of ergastoplasm. The present paper reports the results of an autoradiographic study of DNA generation of such cells. Some experiments were carried out using daily injections of the racemic form of I into rats and a single application of tritiated thymidine and leucine before examination; in others, a single injection of I labelled with tritium was used and tissues autoradiographed after 90 min. o and L forms of I appear to have the same effect, both being incorporated by the same mechanism. The authors support the opinion of Magee & Farber (Cited in F.C.T. 1963, 1, 157) that I, like dimethylnitrosamine, affects cytoplasmic RNA by alkylating it. This produces greatly intensified RNA generation by the micleus which, nevertheless, is unable to compensate the loss sustained through the action of I. Further confirmation for this is also a rapid onset of an inhibition of amino acid incorporation by cytoplasmic proteins which depend on the presence of messenger- and soluble-RNA's. 533. The thyroid suppressed Saxena, K. M., Chapman, E. M. & Pryles, C. V. (1963). Minimal dosage of iodide required to suppress uptake of iodine-131 by normal thyroid. Science 138, 430. Suppression of thyroid function in children is important as a means of preventing uptake of iodine-131 (m I) from nuclear fallout. This end may most readily be achieved by administering sodium iodide (NaI). The object of the present study was to ascertain the minimal dosage needed for this purpose. Children in the age groups 1-3, 4-6 and 9-11 yr were given 100, 300, 600 or 1000 #g NaI daily in aqueous solution. The 24-hr uptake of mI was the main criterion of effect. In 2-yr-old children given 300 gg NaI daily over 8 weeks and 600/~g daily for the next 4 weeks, there was a fall in 13q-uptake. When NaI was discontinued recovery of thyroid function took place within a week and was complete in 2 weeks. It was concluded that, in order to achieve full suppression of thyroid intake of 131I, the minimal effective dose would have to be 1.5-2 mg/m2 of body surface per day or 1-2 mg NaI daily for a child and 3-4 mg for adults. With such doses 50 ~o suppression was achieved in 24 hr and 95 ~ in 4-6 weeks. It is stated on the basis of clinical experience that 100 mg NaI/day given to children over a course of years was without observable toxic effect. It takes several times this dose, continued for years, to induce thyroid goitre. [A determination of the maximum no-effect levels of iodide in children and man is of direct interest to us in view of the liberation of iodide from Erythrosine (Cited in F.C.T. 1963, 1, 268).
291
The 'Skinner box' provides a method of studying the effects of drugs and toxic agents on the behaviour of pigeons. They are trained to perform a task, namely to peck at a key, in order to obtain food. Quantitative criteria of behaviour are provided by the indices of performance, such as number of pecks and frequency of pecking. In fact, with trained birds one obtains records over many months characterized by remarkable precision and reproducibility. A very suitable test material to superimpose on such a background is mercury (Hg) vapour, chronic inhalation of which can lead to the development of tremors, altered behaviour and psychic disturbances in man. Each trained pigeon was exposed in a chamber to an average concentration of 17 mg Hg/m 3 for 2 hr daily, 5 days a week either until it failed to respond or else showed a markedly lower rate of response for 1-3 days; or until it died. When exposure to Hg ceased, the bird was considered to have recovered when the overall response rate proved constant for several weeks. All birds manifested a decrease in response rate, but the time of onset of this effect varied from one to another. In one bird the rate dropped to under 50 % of normal after I week's exposure, rose and fell again in week 4. In 4 pigeons the fall to below 50% took place in week 2 while in 2 others 5 and 6 weeks of exposure were needed. In one hardy warrior the level remained above 50% for 12 weeks. Yet recovery after cessation of exposure bore no relation to the duration of Hg inhalation. The first gross sign of Hg poisoning was tremor, appearing at 5 weeks and becoming more evident during preening, walking and feeding. Weight loss was a late development in birds whose response rates had fallen significantly earlier in the experiment. Analysis of the behavioural effects revealed an early central nervous stimulant action in some birds, but depression was the most prominent general effect. The parallel with human toxic effects of Hg extended to a similar failure to react to environmental stimuli. Although the Maximal Acceptable Concentration for human exposure is 0-1 mg Hg/m 3, exposure of the birds to 17 mg Hg/m 3 failed to produce histological evidence of tissue damage by Hg in the pigeons that died. 531. From cell to whole animal
Smith, C. G., Grady, J. E. & Northam, J. I. (1963). Relationship between cytotoxicity in vitro and whole animal toxicity. Cancer chemother. Rep. No. 30, 9. When a chemical is administered to an animal a very large number of extraneous factors tend to influence the observed biological effect, so that the final outcome may be no reflection of the 'innate' toxicity of the material on living cells. The engaging idea presents itself: why not apply the test material directly to living cells, for example the tissue culture ? This approach assumes even greater importance when we recall that human cell lines, such as liver, can now be maintained successfully in vitro. The obvious difficulty is that cells grown in this way are delicate creatures at the best of times and to wipe them out is not nearly as difficult as to maintain them successfully. Any attempt to correlate cytotoxicity, i.e. inhibition of cell growth or IDs0, with acute LDs0 is therefore of great interest. The authors went further. Since they were dealing with compounds screened for possible use as cancer chemotherapeutic agents they were also able to compare laboratory findings with the dose that proved clinically useful in man. The outcome of the exercise was a statistically significant correlation between inhibition of growth of mammalian cells in tissue culture and acute intraperitoneal toxicity to mice, as well as a correlation with the maximum tolerated dose for man. For any individual compound, however, cytotoxicity was not a useful guide to its behaviour in mice and man. In fact there were some startling discrepancies--for example, fluoroacetate and a barbiturate were innocuous at high levels to cells in vitro; the former has an acute LD50 in mice of 10
292
BIOCHEMICAL PHARMACOLOGY
mg/kg and an estimated oral LDs0 in man of 15 mg/kg. Equally interesting were the IDs0 and LDs0 values (given in that order) for the following pairs of compounds: dimethylformamide >5000, 650; dimethylacetamide >5000, 3000; similarly fumaric acid >1000, 200; 2,4-dich'.orophenoxyacetie acid 45, 838.
BIOCHEMICAL PHARMACOLOGY 532. Mode of action of ethionine Oehlert, W., Kramseh, D. & Beck, V. (1963). Der Einfluss des Abbaues cytoplasmatischer Strukturen auf die DNS-Neubildung bei der experimentellen Athioninvergiftung. Naturwissenschaften 50, 307. Poisoning by ethionine (I), the ethyl homologue of methionine, is known to produce loss of cytoplasmic nucleotides in cells with a high protein metabolism; this also results in decorttposition of ergastoplasm. The present paper reports the results of an autoradiographic study of DNA generation of such cells. Some experiments were carried out using daily injections of the racemic form of I into rats and a single application of tritiated thymidine and leucine before examination; in others, a single injection of I labelled with tritium was used and tissues autoradiographed after 90 min. o and L forms of I appear to have the same effect, both being incorporated by the same mechanism. The authors support the opinion of Magee & Farber (Cited in F.C.T. 1963, 1, 157) that I, like dimethylnitrosamine, affects cytoplasmic RNA by alkylating it. This produces greatly intensified RNA generation by the micleus which, nevertheless, is unable to compensate the loss sustained through the action of I. Further confirmation for this is also a rapid onset of an inhibition of amino acid incorporation by cytoplasmic proteins which depend on the presence of messenger- and soluble-RNA's. 533. The thyroid suppressed Saxena, K. M., Chapman, E. M. & Pryles, C. V. (1963). Minimal dosage of iodide required to suppress uptake of iodine-131 by normal thyroid. Science 138, 430. Suppression of thyroid function in children is important as a means of preventing uptake of iodine-131 (m I) from nuclear fallout. This end may most readily be achieved by administering sodium iodide (NaI). The object of the present study was to ascertain the minimal dosage needed for this purpose. Children in the age groups 1-3, 4-6 and 9-11 yr were given 100, 300, 600 or 1000 #g NaI daily in aqueous solution. The 24-hr uptake of mI was the main criterion of effect. In 2-yr-old children given 300 gg NaI daily over 8 weeks and 600/~g daily for the next 4 weeks, there was a fall in 13q-uptake. When NaI was discontinued recovery of thyroid function took place within a week and was complete in 2 weeks. It was concluded that, in order to achieve full suppression of thyroid intake of 131I, the minimal effective dose would have to be 1.5-2 mg/m2 of body surface per day or 1-2 mg NaI daily for a child and 3-4 mg for adults. With such doses 50 ~o suppression was achieved in 24 hr and 95 ~ in 4-6 weeks. It is stated on the basis of clinical experience that 100 mg NaI/day given to children over a course of years was without observable toxic effect. It takes several times this dose, continued for years, to induce thyroid goitre. [A determination of the maximum no-effect levels of iodide in children and man is of direct interest to us in view of the liberation of iodide from Erythrosine (Cited in F.C.T. 1963, 1, 268).