David A. Karnofsky

THE EFFECTS OF METALS ON THE CHICK EMBRYO: TOXICITY AND PRODUCTION OF ABNORMALITIES IN DEVELOPMENT

The effects of the elements were studied principally in the fourand eight-day-old embryo since developmental abnormalities are often grossly evident a t these older ages. Furthermore, elements generally toxic to embryos early in development may produce selective growth effects when given to more differentiated embryos. Elements found to produce specific and consistent abnormalities when injected at four or eight days were studied further in younger and older embryos. Commercially supplied White Leghorn eggs were used. They were maintained a t a temperature of 38C, and candled daily for viability. The volume of the test agent injected per egg ranged from .05 cc. to 0.2 cc. with an occasional yolk sac injection of 0.3 or 0.4 cc. When possible, water soluble salts were used, but, in rare cases, insoluble salts were ground in distilled water and injected as a suspension. The injections were made by two routes: into the yolk, or onto the chorioallantoic membrane (CAM) of eight-day eggs. Yolk sac injections were made through a hole drilled in the blunt end of the egg, and the opening then was sealed with melted paraffin. The CAM was injected through a small window cut in the shell directly over the membrane and the opening was sealed with Scotch tape after the injection. The preliminary toxicity tests were carried out in the four-day-old embryo. Four eggs were injected at each of three of four dose levels, and, after several trials, the LDbo dose was approximated. Groups of six to ten eggs were injected at several dose levels above and below the estimated L D ~ o in order to obtain a more precise figure. In many instances, several confirmatory toxicity runs were necessary to obtain a reasonably consistent LD60 figure. With the four-day yolk sac data as a guide, similar toxicity studies were completed in the eight-day embryo by the yolk sac and CAM routes. The embryos were candled daily and, each day, the dead embryos were recorded and examined. Occasionally, embryos were sacrificed for specific

Immunologic factors and resistance to infection in chronic lymphatic leukemia.

Abstract Twenty-two patients with chronic lymphatic leukemia were studied clinically, hematologically and immunologically. The following immunologic tests were performed: serum electrophoresis, isohemagglutinins, response to typhoid-paratyphoid vaccination and to tuberculin, mumps and C. albicans skin tests, complement and properdin titers, and determination of serum macroglobulins. The patients were divided into two groups according to their history of infectious disease. Patients with chronic lymphatic leukemia who had frequent infectious complications could, in general, be distinguished by their lower serum gamma globulin levels. None of the other parameters considered allowed us to differentiate between the two groups. No relationship was found between duration of disease and hypogammaglobulinemia in this series, but there was some indication that hypogammaglobulinemia was a late stage in the natural history of chronic lymphatic leukemia. The various serum protein abnormalities which may characterize patients with this disease suggest that despite morphologic similarities there may be more than one kind of chronic lymphatic leukemia.

Effects of Large Doses of Chloramphenicol on Human Subjects

WEN chloramphenicol first became available for general use in 1948, as one of the earliest broad-spectrum antibiotics, it was regarded as an important contribution to the treatment of infectious di...

Quinacrine (Atabrine®) in the Treatment of Neoplastic Effusions

Excerpt Quinacrine (Atabrine®) inhibited the growth of several transplanted solid tumors (1) and of the Ehrlich ascites tumor in mice (when it was given intraperitoneally) (2). It was cytotoxic to ...

Effect of Folic Acid, “4-Amino” Folic Acids and Related Substances on Growth of Chick Embryo.

Summary The “4-amino” pteroylglutamic acids profoundly inhibit the growth of the chick embryo, with the production of developmental abnormalities. These compounds are active in the range of 0.003 to 0.005 mg/egg, and their actions are not prevented by large doses of folic acid. The “4-amino” folic acids with aspartic acid, threonine and alanine substituted for the glutamic acid are considerably less toxic but produce similar toxicological effects. Other compounds allied to folic acid are relatively non-toxic and do not possess the growth-inhibitory activity of the “4-amino” compounds. 2,6-diaminopurine, at LD30 doses, does not seem to be an active growth inhibitor, but it produces a pale embryo, presumably deficient in hemoglobin.

Serial passage of human tumors in chick embryo: growth inhibition by nitrogen mustard.

Summary Human Sarcoma #1 (H. S. #1), Human Epidermoid Carcinoma #3 (H. Ep. #3) and Human Embryonal Rhabdomyosarcoma #1 (H. Emb. Rh. #1) have been grown by serial passage on the chorioallantoic membrane of the chick embryo, and the H.S. #1 and H. Ep. #3 by serial passage in the yolk sac. Metastases of the H. Ep. #3 to the liver and kidneys of the host chick have been observed. The H. S. #1 and H. Ep. #3, when treated with a nitrogen mustard, HN2, showed the same pattern of histological injury previously described for the mouse sarcoma 180 growing in chick embryos and mice.

Tumor transplantation to the chick embryo.

In 1912, J. B. Murphy reported that mouse and rat tumors would grow when explanted to the chorio-allantoic membrane (CAM) of the chick embryo, and that these tumors could be maintained by continuous passage from egg to egg. The year previously, Rous and Murphy2 demonstrated that the Rous chicken sarcoma, transplanted to the CAM, would induce tumors of the membrane. Since these original reports, tumors from chickens, ducks, mice, rats, rabbits, guinea pigs and man have been explanted in the fertile egg, by the CAM, yolk sac, intra-embryonic and intravenous routes. The developing chick embryo has no defense against the growth of heterologous tissue, and it will supply blood vessels and connective tissue for the growth of these tumors. The following methods are examples of different procedures used in tumor transplantation: Chorio-allantoic Membrane Route.3 The eight-day embryo is used. A window is cut in the shell over the vascular area, and a tumor fragment, 1-2 mm. in size, is placed on a large vessel. The shell opening is sealed with Scotch tape, or with a coverglass and paraffin. The egg is incubated a t 99100 C.,4 and the embryo is sacrificed a t 17 to 18 days, and the tumor removed. Yolk Sac R o ~ t e . ~ The four-day embryo is used. Tumor tissue is prepared by adding one cc. of tumor suspension to four cc. of a 1: 1 mixture of egg white and saline. One-half cc. of this tumor preparation is injected into the yolk sac, and the embryos sacrificed a t 17 days. Intravenous Route.6 The 11-day embryo is used. The shell is removed over the air sac and the blood vessels exposed. Five-hundredths (0.05 cc.) of the sterile tumor suspension is injected intravenously, and the embryo is sacrificed on the 20th day. Intra-embryonic Route.6 Embryos 50-60 hours old (26-34 somites) are used. The embryo is exposed through a window in the shell, and a strip of somatopleure lateral to somites 24 to 32 is removed. A piece of tumor tissue is fitted into place, and the shell opening repaired. The embryos are usually sacrificed six to eight days later. The behavior of tumors explanted to the chick embryo has been measured by the growth of the tumor, its histological appearance, growth by continuous passage in eggs, viability when transplanted to its original host, and the effect of the growing tumor on the chick embryo. The chick embryo has been used in many tumor studies, and we will summarize some of this work with illustrative material drawn from our own studies.

Chemotherapy of Hodgkin's disease

Radiotherapy has the first priority in planning an appropriate therapeutic program for the patient with Hodgkins disease but chemotherapy also has acquired an important role. Drugs which have demonstrated therapeutic activity in Hodgkins disease include: polyfunctional alkylating agents, vinca alkaloids, N‐methylhydrazine, adrenal steroids and actinomycin D. The antimetabolites have been less successful; an irregular and brief effect is reported for methotrexate and generally no benefit at all from 6‐mercaptopurine and 5‐fluorouracil. The indications for drug therapy include: (1) localized Hodgkins disease in combination with x‐ray therapy; (2) in acute situations when rapid relief of signs or symptoms are necessary and (3) for widespread disease with systemic manifestations. There are no certain guides to the choice of drug or regimen and the therapeutic program in each case must be adapted to the patient and to the treatment modalities available.

FOLIC ACID ANALOGS AND EXPERIMENTAL TUMORS

Earlier papers in this monograph have reviewed the classic studies on antimetabolites and reported recent extensions2 of this profitable concept. Approximately three years ago, the extensive, effective studies demonstrating the structure of folic acid and some of its biological activities were reported in a symposium of the Academy? It was a natural consequence that analogs of folic acid should be prepared and studied for both folic and antifolic acid activity. Many of the subsequent findings in bacteria and animals have been reported in this monograph !-g In view of the essential role of folic acid in the growth of certain cells, it was logical that the folic acid antagonists be studied for adverse effects upon tumor tissue. It could have been predicted from the studies of folic acid deficiencies that compounds possessing antifolic acid activity would not be without adverse effects upon normal mammalian cells. Jukes el aL6 and Philips et aL9 have just reported studies indicating the nature of toxic effects of 4-amino pteroylglutamic acid and its congeners in mammals. It was with much of this background that studies on folic acid analogs were initiated in the hope that compounds would be found in which the toxicity to abnormal cells would be much greater than that for the most susceptible normal cells. For the past year, the Division of Experimental Chemotherapy of the Sloan-Kettering Institute has concentrated study upon nearly 300 analogs of folic acid and simpler related compounds such as the pteridines, pyrimidines, and purines in a search for adverse effects upon abnormal tissue in a number of experimental conditions. This paper will be confined to studies of experimental animal tumors even though some of the compounds have reached clinical trial in a number of The folic acid antagonists included in this discussion are 4-amino pteroylglutamic acid, 4-amino-NlO-methyl pteroylglutamic acid, 4-amino pteroylaspartic acid, and 2,ti-diaminopurine (FIGURE 1). After the compounds are filed and coded for future reference, they may go directly for test against mouse tumors in tissue culture or in egg culture. Prior to the study against leukemia and Sarcoma 180 in mice, the compounds are submitted to the Pharmacology Section for determination of toxicity. Compounds of sufficient interest are studied in all of the tests and also submitted to trial against a spectrum of tumors in mice and rats. Compounds Compounds have been received from a number of s0urces.t

Effects of physiological purines on the development of the chick embryo

Abstract The commonly occurring purine bases, ribonucleosides and deoxyribonucleosides were injected into the yolk sac of the 4-day-old chick embryo to determine their toxicity and teratogenic activity. The deoxyribonucleosides were most toxic, with dGuR, dAdR and dHxR having estimated LD 50 s of 2, 4 and 4 to 8 μmoles/egg, respectively. The deoxyribosyi derivatives caused marked disturbances in development of the 4-day embryo, with stunting of growth, edema, facial defects, absent bones and shortened extremities. dGuR was most active in producing weight inhibition and severe developmental disturbances; these effects were seen at 1 μmole/egg for dGuR, in contrast to 16 μmoles/egg for dHxR, and at an excess of 8 μmoles/egg for dAdR.