We have found that if suitable concentrations of toxin are employed a rapid and reproducible neuro-muscular block can be produced in the isolated rat phrenic nerve-diaphragm preparation of Bulbring (1946). This preparation is adaptable and has proved very suitable for the analysis of the action of the toxin. Torda & Wolff (1947) have claimed that choline acetylase is strongly inhibited by the toxin and that this might explain how the toxin produces neuromuscular block. The action of the toxin on this system and on the closely related aromatic amine acetylase has been investigated further. METHODSToxin. Type A toxin was obtained from Clostridium botulinum (strain 4587) grown in a medium consisting of 25% tryptic digest of casein, 10% yeast extract, and 1 % glucose. The ingredients were prepared as in CCY medium (Gladstone & Fildes, 1940). The toxin was purified by acid precipitation with HCl at pH 3 9, then rendered soluble by elution with 0-2M-Na2HPO4 to pH 6-0.The solution was freeze-dried and stored as a powder in vacuo over phosphorus pentoxide. As required, the toxin was dissolved in a buffer consisting of 1% Na2HPO4 and 0-2 % gelatin at pH 6-6 to give a concentration of 2 x 105 or 2 x 106 mouse LD50/ml. . (Lamanna, McElroy & Eklund, 1946), i.e. this material was c. 5% pure toxin.) In the text the amount of toxin used is expressed in terms of mouse LDr0/ml. of bath fluid which is for convenience called a unit.
THE distribution of citric acid in the animal body has not previously been systematically studied, for although Thunberg and his colleagues have made a careful examination of the citrate content of body fluids, that of the solid tissues has been largely neglected. This is all the more surprising, since Pucher et al. [1936] have described a sensitive colorimetric method which, unlike the enzymic one of Thunberg, is readily applicable to such material. Pucher et al. [1936] and Sherman et al. [1936] analysed only a few tissues by the colorimetric method (liver, kidney, abdominal and heart muscle of the dog), for which very low values were found (0-119 mg. citric acid/100 g. tissue). It is clear from their paper that they regarded these results as typical of the major tissues of the body, but this view is shown to be incorrect by the present work.In-contrast with these low results on solid tissues, it has long been known that several body fluids contain relatively large amounts of citrate. Thus milk contains enough for the crystallization of Ca citrate from evaporated milk, an occurrence which led to the first identification of citric acid as a normal constituent of the animal organism by Soxhlet &iHenkel [1888]. Citric acid was not isolated from urine until many years later [Fasold, 1930], although it was shown to be a normal constituent by Amberg & McClure [1917], and in fact urine may also contain amounts of citric acid of similar order to that present in milk mg./100 ml.; for literature see Ostberg [1931], Schersten [1936], Gronvall [1937], Sherman et al. [1936]). According to the Scandinavian workers, much less citric acid is present in cerebrospinal fluid, amniotic and follicular fluids, blood serum, aqueous humour, saliva and sweat, which contain in diminishing order about 50 1 mg./100 ml. Pucher et al. [19311] found similar low figures for blood and saliva. Finally there is the surprising observation of Schersten [1929, 1936] that a high concentration of citric acid (up to 410 mg./100 ml.) is present in semen, and is particularly associated with the secretion of the seminal vesicles, which may contain as much as 633 mg./100 g. This brief outline of the present knowledge of citric acid distribution may be supplemented by the comprehensive reviews already quoted. The present investigation is primarily the outcome of analyses of tumour tissue, a material of which the citric acid content has not previously been studied. The results showed that tumour frequently, though not invariably, contains a relatively high concentration of citric acid. However, certain normal organs 1 The greater part of this work was reported to the Biochemical Society at Sheffield in Feb. 1940 [Dickens, 1940]. Its completion was delayed by difficulty in obtaining necessary replacements for the photometer used.
ALTHOUGH such a wide variety of chemical substances has beeil tested for carcinogenic activity, the group of lactones has been largely neglected. Thus, Hartwell (1951) and Shubik and Hartwell (1957) together list 1329 plus 779, a total of 2108, different compounds tested, but very few lactones appear in their comprehensive Survey, and in almost all instances even these have Ilot been tested over any adequate period for their possible carcinogenic action to have been detected.One of the very few lactones hitherto satisfactorily studied in this respect is /J-propiolactone. This vesicant substance was found to be mutagenic in Neurospora by Smith and Srb (1951), a finding which led Walpole et al. (1954) to study the effect of prolonged subcutaneous injections extending over 13 weeks into rats of /I-propiolactone, 2 mg./100 g. body weight, dissolved in arachis oil. Of 12 rats so treated, 9 developed sarcomas at the injection site after 28 to 55 weeks from the start of administration. Roe and Salaman (1955) decided that for the skin of the mouse /&-propiolactone was undoubtedly an " initiator " in carcinogenesis in the sense used by Friedewald and Rous (1944) giving tumours rapidly when alternate treatments with croton oil as " promoter " were also given. At first they expressed doubt whether /3-propiolactone is itself carcinogenic for the skin of the mouse, but this was apparently due to too short a period of observation, since Roe and Glendenning (1956) later observed tumours after 27-52 weekly paintings of 2 5 per cent /J-propiolactone in acetone (papillomata arose in 5 mice of 9 treated, becoming malignant in 2 mice after 40 weeks applications). With higher initial doses, the early ulceration and scarring produced were followed by earlier malignant change (3 carcinomas in 20 mice after 21 weeks). Consequently /J-propiolactone must be considered definitely a carcinogenic substance, whether given subcutaneously to rats or by application to the skin in mice. In either case repeated applications are necessary for tumour production; fewer applications than those given above have failed to produce tumours in mice (Salaman, 1959), and this is also shown in the present paper to apply to subcutaneous injections in the rat. This negative result was in fact to be anticipated from the short period of persistence of the agent, due to the ease of breakdown of this unstable lactone ring in the body (cf. Dickens, Jones and Williamson, 1956). This also applies to a varying extent to the other lactones whose carcinogenic properties are described in this paper, all of which were therefore applied repeatedly over long periods, and usually from an oily depot to assist in slow liberation and elimination.
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