In the normal rat given a single dose of one mg N,N-dimethyl-4-aminoazobenzene (DAB) via the hepatic portal vein the following biliary metabolites reached their maximal rates of excretion in the sequence: 4'-sulphonyloxy-DAB, N-(glutathione-S-methylene)-4-aminoazobenzene (GSCH2AB), 4'-sulphonyloxy-N-methyl-4-aminoazobenzene (4'-sulphonyloxy-MAB) 4'-sulphonyloxy-GSCH2AB and MAB-4'-beta-glucuronide. The unusual and relatively unstable N-methylene glutathione conjugates were major metabolites accounting for up to 70% of the whole. It was shown that all the 4-aminoazobenzene (AB) and perhaps all of the 4'-sulphonyloxy-AB, which may be observed in bile, are artefacts due to decomposition of GSCH2AB and 4'-sulphonyloxy-GSCH2AB respectively and that biliary excretion of N-methyl oxidised products of MAB and 4'-hydroxy-MAB is dependent on their conversion to the GSH conjugates, GSCH2AB and 4'-hydroxy-GSCH2AB respectively. Sulphotransferase inhibition by pentachlorophenol caused a reduction in the excretion of all sulphate conjugates, but biliary excretion as a whole was not reduced significantly due to a compensatory increase in the excretion of MAB-4'-beta-glucuronide and the appearance of 4'-OH-GSCH2AB. Glutathione (GSH) depletion by diethylmaleate caused a reduction in biliary metabolites of DAB by lowering the levels of GSH conjugates. This was because the amount of N-methyl oxidation of MAB and 4'-hydroxy-MAB were proportional to the amount of GSH present. The fall in N-methyl oxidation was not compensated for by an increase in 4'-hydroxylation and was accompanied by a delay in the appearance of 4'-hydroxylated metabolites. The administration of potential precursors of 4'-sulphonyloxy-GSCH2AB establishes the sequence of reactions resulting in its formation to be 4'-hydroxylation, N-methyl oxidation, GSH conjugation and O-sulphation.

Evidence was obtained that the response of blood lymphocytes to phytohemagglutinin was reduced in some but not all rats with massive hereditary renal tumors (less than 50 mm mean diameter). Erythrocyte-antibody sensitized, complement dependent (EAC) rosette formation by sensitized spleen cells was also depressed in these animals, but plaque forming cell activity was essentially normal. Lesser tumor growth was associated with general cell-mediated and humoral immune responses which were within the range found for non-tumor-bearing syngeneic rats.

Further histological findings for the naturally occurring kidney tumor of the Eker rat included the unusual location of the tumor entirely within the medulla rather than in the more usual site within the kidney cortex; the presence of psammoma bodies, and metastases of the tumor in one animal. The kidney tumors were shown to be transplantable with a latent period of about nine months. Histologically, the transplanted tumor, even at the twenty-eight transplant generation, maintained the cellular characteristics of the primary. The histological and transplant data allow the classification of the definitive tumor as a primary adenocarcinoma. The suitability of the tumor as an animal model for human renal carcinoma is suggested.

A Porton and a hooded rat strain showed a raised LD50 for dimethylnitrosamine (DMN) when pre-conditioned on a protein-free and/or a sugar diet. Little or no such differential toxicity between normal and diet-fed animals was found for rats of the Wistar, BDIX and CFY strains. Pre-treatment with CCl4 did not alter significantly the toxicity of DMN in the Wistar strain. All 5 rat strains treated by diet or CCl4 administration metabolized DMN at a very much slower rate than did the controls, the rates for the different strains being quantitatively similar. It is concluded that the toxicity of DMN is not necessarily related to its rate of metabolism and that the effect of diet or CCl4 treatment of DMN toxicity is dependent on the strain of rat used.

An apparatus easily manufactured in the laboratory is described for the continuous infusion of substances into the stomach of the unanaesthetised rat.

N-Nitroso-N-methylurea (NMU) induced a marked dose dependent leucopoenia which was associated with an increased survival of skin allografts in adult rats and in 2 strains of mice. The humoral immune response to NMU as assessed by haemolytic plaque formation and haemagglutination was also much reduced. Dimethylnitrosamine (DMN) which, like NMU is a powerful carcinogen and an alkylating agent, showed no immunosuppressive activity after a single dose in rats on either a normal diet or fed a protein-free diet which enhances kidney tumourigenesis. In mice DMN at a near LD(50) dose (14 mg/kg) had no effect on skin graft survival but did reduce the humoral response. At half this dose level, however, no immunosuppressive effect was seen. The results support the conclusion that the immunosuppressive activity of a chemical carcinogen is not necessarily associated with the expression of its carcinogenicity.

N-Nitroso-N-methylurea (NMU) at a dose of 50 mg/kg body wt significantly increased the incidence of early foetal death (dominant lethal mutations) when compared with the solvent controls in the mouse dominant lethal test. The mutagenic activity of NMU was considerably less than that of methyl methanesulphonate (MMS) which was used as a positive control mutagen but, in comparison, it affected a greater part of the spermatogenic cycle of the treated male mice. N-Methyl-N-nitroso-N′-nitroguanidine (MNNG) produced a small increase in dominant lethal mutations with a low level of significance (P < 0.05) at doses of 50 and 70 mg/kg body wt during the first week only of the 8-week assay period. A highly significant, though still moderately increased response was found for the near-LD50 dose of 100 mg/kg. At this dose level the mutagenic activity was confined to the first 4 days of the first week. N-Nitrosomorpholine could not be tested during the first 3 to 4 weeks of the assay at dose levels of 50 and 100 mg/kg due to an adverse effect on mating activity but no increase in the incidence of dominant lethal mutations was observed between the 5th and 8th week. At a dose of 35 mg/kg mating activity was normal during the initial 3-week period studied but there was no evidence of increased mutations.

ABSTRACT Rats hypophysectomised on Day 8 of pregnancy showed no growth or involution of the corpus luteum four days later but there was a significant increase in the total and free acid phosphatase activities. Hypophysectomy on Day 12 did not affect the expected changes in luteal weight and acid phosphatase activity but did increase the volume of the corpus luteum on Day 16 compared to intact pregnant rats. Concurrently performed hypophysectomy and Caesarian section caused a greater rate of luteal regression and a greater increase in acid phosphatase activity than did Caesarian section alone. Although involution of the corpus luteum was progressively advanced by 32 days after the initiation of gestation in rats hypophysectomised or hypophysectomised plus Caesarian sectioned on Day 8 and 12 respectively, there was no proportional increase in the acid phosphatase activity. The role of acid phosphatase in the structural regression of the rat corpus luteum is considered to be minimal.

A simple and rapid technique for intravenous injection into the sub-lingual veins of common laboratory rodents is described. No prior preparation other than light anaesthesia is required and repeated injections can be made into the same vein.

IT has now been possible to test 33 compounds with a lactonic or related structure, of which 25 have proved on subcutaneous injection to be capable of inducing the formation of tumours of the connective tissue elements of rats (Dickens and Jones, 1961; 1963a, b; 1965).The carcinogenic substances are for the most part characterised by chemical structures which indicate their reactive nature.This may be due to: a. Strain in the lactone ring, particularly in the 4-membered rings.b.Conjugation of double bonds, within and outside the ring, with the lactonic carbonyl group.c.Conjugation of double bonds even in linear compounds.d.Presence of the anhydride ring of dicarboxylic acids.Wlhere these features were absent, or where the substances caused death of the animals before tumours could be expected to appear, it was not possible to show a carcinogenic action in members of this series.Dickens and Cooke (1965) studied the rates of hydrolysis and of interaction with cysteine of these substances and showed that, with the exception of aflatoxin and N-ethyl maleimide, the rates of reaction with cysteine could be correlated in a very approximate manner with their carcinogenic activity.It is becoming clear that carcinogens belonging to this chemical class might be a pertinent factor in human cancer.Many compounds of this general type are widespread in animal and vegetable tissues, are produced by some saprophytic fungi infecting foodstuffs, and may possibly be produced during the burning of tobacco (Dickens, 1963).Some of them are useful antibiotics, and it is unfortunate that the very molecular structure which makes them active agents against bacteria and fungi is probably also that which makes them active as carcinogens.In relation to this problem it is important to know whether such compounds are carcinogenic not only when injected but when taken into the stomach and lungs, these being the routes by which some substances related to those dealt with in these studies are liable to be ingested by man.This paper reports the results of testing four further substances chosen because of the similarity of their structure to other compounds which have already proved to be carcinogenic.They are sorbic acid and dehydroacetic acid-salts of which * For structural formulae I, II, III and IV see p. 140.