Thymomas induced by simple alkylating agents in C57BL/Cbi mice: kinetics of the dose response.

Specific-pathogen-free inbred C57BL/Cbi mice (adult virgin females) were given single sublethal doses of N-methyl-N-nitrosourea or N-ethyl-N-nitrosourea and were studied for a lifetime for the development of thymomas. A fatal lymphocytic lymphoma with a "starry sky" pattern due to the presence of macrophages was induced in the thymuses of treated mice within 250 days of treatment. Control and low-dose treatment groups had up to 70% incidence of a histiocytic lymphoma that was usually primary in mesenteric lymph nodes and nearly always occurred later than 250 days after treatment. A "one-hit" linear relationship existed between the time of appearance of induced thymic lymphomas and the log fraction of non-tumor-bearing mice. The absolute latency period of these tumors was constant and independent of dose. The effect of dose was an exponential increase of the total incidence of induced thymic lymphomas. By mathematical analysis, the best estimate of the exponent from the results was 2 or 3, indicating that the development of these induced tumors may be produced by 2 or 3 "events" in the target cell. Possible candidates for these events are premutagenic alkylation of DNA, inactivation of DNA repair, oncovirus activation, regenerative hyperplasia, development of trisomy No 15, and inhibited immunosurveillance.

Lower limb paralysis induced in mice by a temperature-sensitive mutant of Moloney leukemia virus.

A temperature-sensitive mutant of Moloney murine leukemia virus defective in an early function and injected into newborn mice produced lower limb paralysis. Susceptible mice were inbred strains CFW/D, CBA/H, C3H/Bi/Ka, and outbred NIH Swiss stock. Inbred W/Fu rats and C57BL/Ka mice did not develop the paralysis, though the latter were infected with virus; the sera from these mice produced paralysis in susceptible CFW mice.

Synthesis of 1-(2-hydroxyethyl)-1-nitrosourea and comparison of its carcinogenicity with that of 1-ethyl-1-nitrosourea.

1-(2-Hydroxyethyl)-1-nitrosourea (HNU) was prepared by the action of nitrosyl chloride on (2-hydroxyethyl)urea. Attempts to synthesize HNU by an earlier described method were unsuccessful and led to the formation of the cyclized derivative 1-nitroso-2-oxazolidone. In addition, the spectral data that we obtained for HNU differed from those reported earlier. Female C57BL/Cbl mice were treated with single ip doses of HNU to determine its median lethal dose (LD50) and its ability to induce lymphocytic thymic lymphomas in these mice. The results showed that the LD50 was the same as that for 1-ethyl-1-nitrosourea (ENU) and that its was slightly more potent than ENU as a carcinogen in this system.

Endoscopic large bowel polypectomy. Adequate treatment of some completely removed, minimally invasive lesions.

Eighteen large bowel adenomas with invasion of the head or the stalk were removed by endoscopic polypectomy or by segmental resection at University Hospital, London, Ontario between 1973 and 1982. All were completely removed by histological criteria. All the patients were traced for an average follow-up period of 4.6 years. None had developed disseminated large bowel cancer. Adding these results to those in the literature, it appears that, provided there is not a high degree of anaplasia of the tumor or lymphatic or venous invasion, endoscopic polypectomy is adequate therapy for such adenomas. An endoscopic recheck of the site of removal after approximately 2 months may be worthwhile as a few local recurrences of benign tumor were reported, although not in the present series. The patients should be followed for life.

Medical audit of rectal biopsy diagnosis of inflammatory bowel disease.

The records of the rectal biopsy diagnoses of ulcerative colitis and Crohn's disease in the Department of Pathology, St Mark's Hospital, London, were reviewed. The biopsy diagnoses were compared to subsequent resection diagnoses on the same patients, and annual and seasonal variations in the frequency of these and related diagnoses were studied. The accuracy rate for the biopsy diagnosis of ulcerative colitis was about 70% and for Crohn's disease about 40% each time a biopsy was read. The low figure for the accuracy rate for Crohn's disease could be attributed to sampling error inherent in the diagnosis of a disease which is essentially patchy, showing discontinuous pathology. Also, many patients with Crohn's disease have a normal rectum which is biopsied to demonstrate the distinction from ulcerative colitis. In practical terms therefore a 40% accuracy rate in Crohn's disease is probably adequate. The rate of "false-positive" diagnoses was about 5%. There was a seasonal variation in the frequency of these two diagnoses, but no variation attributable to changes in observers, as pathology trainees in the Department change regularly. The frequency of diagnoses of non-specific inflammation and of normal colon did show such non-random variations.

Cytogenetics of murine thymic lymphomas induced by N-methyl-N-nitrosourea: difference in incidence of trisomy 15 in thymic lymphomas induced in neonatal and adult mice.

Chromosome complements of murine thymic lymphomas induced by an alkylating agent N-methyl-N-nitrosourea (MNUA) were analyzed microscopically and karyotypically using the Q-banding technique. The chemical carcinogen was injected intraperitoneally into either neonatal or 7-week-old CFW/D mice. In addition, thymic lymphomas induced in 7-week-old AKR mice and thymic lymphomas developed spontaneously in this strain were also examined. All six lymphomas induced in neonatal CFW/D had hyperdiploid cell lines that accounted for 90% of the cells analyzed. Chromosome analysis of lymphomas induced in adult DFW/D mice showed that only out of nine lymphomas had predominantly hyperdiploid cell lines. The remaining five lymphomas had diploid modal chromosome number although they also carried a variant line characterized by 41 chromosomes. All eight lymphomas induced in adult AKR mice and six out of seven spontaneous AKR lymphomas showed predominantly diploid modal line. The remaining spontaneous lymphoma had a hyperdiploid stem line of 41 chromosomes. Microscopic and karyotypic analysis further identified trisomy 15 as the regular chromosome abnormality in the hyperdiploid cells in lymphomas of each group, whereas cells with diploid chromosome number had no detectable chromosome abnormality. Additional trisomies were also found, but their appearance was restricted to individual tumors. Thus, the incidence of trisomy 15 in lymphomas induced by MNUA in adult CFW/D and AKR mice, as well as in the spontaneous AKR lymphomas, is significantly lower than that in lymphomas induced in neonatal mice by the same carcinogen.

Some mechanisms operative in carcinogenesis a review.

Multiple factors contribute to the development of neoplasia. Sometimes a single agency can bring about a tumour if it has many different effects, but at other times a tumour arises more insidiously due to a succession of events [240,241] which by themselves may be innocent. Alterations in the genome of the cell are at the fore-front of our interest because they can be brought about by most of the carcinogenic agents we know. The cell can repair some such alterations but both forward and destructive mutations do appear. The roles of cell proliferation, cell differentiation, the immune mechanism and carcinogen-activating enzymes are beginning to be understood. The effects of dose, route of administration, and of other agents given at the same time [242-245] must not be lost sight of. Other factors no doubt will be added as we begin to look at the structure and function of cell-surface membranes [246-248], at host susceptibility genetics [26, 249], and at the generation of carcinogens inside the body [250,251]. We are only beginning to understand carcinogenesis. In no single instance do we as yet know how a tumour comes about in full details of molecular biology. It is possible that fully rational treatment of cancer will not be possible until we have such an understanding. Once a tumour becomes independent of carcinogenic factors, it continues to develop in a bizarre fashion which makes its study and treatment by all means other than surgery difficult.

Methylation of DNA in various organs of C57B1 mice by a carcinogenic dose of N-methyl-N-nitrosourea and stabiltty of some methylation products up to 18 hours.

Female 6-8-week-old (57B1 mice were injected i.p. with N-methyl-N-nitrosourea (MNUS) (14C or 3H-methyl-labelled) in saline (80 mg/kg) and DNA was isolated from bone marrow, small bowel, kidneys, liver, lungs, spleen and thymus at various times thereafter up to 18 h. Methylation of DNA was found in all organs examined, and by analyses using column or paper chromatography of DNA hydrolysates, the extent of methylation of DNA purines was determined. Methylated guanine residues (at N-3, N-7 and 0-6 positions) were stable in DNA up to 18 h, but methylated adenines (at N-3 or N-7) were removed from DNA of all organs examined; the overall half-life of methyladenines was about 3 h, but removal appeared to occur in a biphasic manner, with a proportion of methyladenine remaining relatively stable. This relative stability was somewhat more marked in bone marrow than in other organs.

Tissue distribution and mode of DNA methylation in mice by methyl methanesulphonate and N-methyl-N' -nitro-N-nitrosoguanidine: lack of thymic lymphoma induction and low extent of methylation of target tissue DNA at 0-6 of guanine.

The methylating agents methyl methanesulphonate (MMS) and N-methyl N'-nitro-N-nitrosoguanidine (MNNG), administered by single i.p. injection in mice failed to yield thymic lymphoma at doses around 60% of the LD50 values, in contrast to MNUA which gives a high yield of tumours by this route. Comparison of the tissue distribution and mode of DNA methylation by these agents showed a positive correlation with ability to methylate the 0-6 atom of guanine in DNA of the target tissues thymus and bone marrow and tumorigeneis. MMS gave a low yield of this product due to its relatively low Sn1 reactivity but was able to methylate DNA extensively at other sites in the target tissues and other organs examined. MNNG despite its ability to methylate 0-6 of guanine in DNA in vitro to the same relative extent as the potent carcinogen MNUA, methylated DNA of thymus and bone marrow to a very small extent in vivo but was able to methylate DNA in certain other tissues nearer the site of i.p. injection. These findings contrast with the general relatively extensive methylation of 0-6 of guanine in DNA of the target tissues and other organs by N-methyl-N-nitrosourea (MNUA).

Chromosome damage in the bone marrow of mice treated with the methylating agents methyl methanesulphonate and N-methyl-N-nitrosourea in the presence or absence of caffeine, and its relationship with thymoma induction.

A single dose (0.8 mmole/kg) of N-methyl-N-nitrosourea (MNUA) causes significantly more chromosome damage in the bone marrow of mice than a dose of equal toxicity to the animals, (1.1 mmole/kg) of methyl methanesulphonate (MMS) 6, 24 and 48 h after treatment. At these doses both agents alkylate bone-marrow DNA to similar extents, but only MNUA induces thymic lymphomata. The greater chromosome-damaging effects of MNUA are ascribed to the known differences in the pattern of DNA alkylation by each agent, in particular the much higher levels of O-6 methylguanine and phosphotriesters produced by MNUA. The greater chromosome-damaging effect of MNUA may account for its higher toxicity to the bone marrow which in turn may be a significant factor in the induction of thymomata. The enhancement by caffeine of chromosome damage seen particularly 48 h after MMS-treatment suggests that post-replication repair protects cells from the effects of DNA-methylation in vivo.

Stable genes.

Some genes such as those for histones and RNAs are conserved unchanged through much of evolution and have numerous tandem repeat copies in the genome. It is proposed that as yet undetected 'polystrand' enzymes use such multiple copies as a means of conserving their sequence by comparing the copies and eliminating errors.

Stable and unstable genes: distribution among species and range of mutations.

An expansion of the 'stable' gene hypothesis proposed previously is the hypothesis that there is a range of genes, some proto-oncogenes and some not, varying from those with multiple copies in the genome, wide distribution among species, and limited mutability because of a specific protective stabilizing mechanism of such genes, to those that are in single copies in the genome, unprotected, unique to one species, and highly mutable along their entire length.