Nitrogen Mustard Treatment Research Articles

Effect of nitrogen mustard of hyaluronate

The effect in vitro of nitrogen mustard (HN2) treatment on physical parameters of partially purified hyaluronate from human umbilical cords and of synovial fluid from patients with rheumatoid arthritis was studied. Intrinsic viscosities, gel filtration elution patterns (Sepharose 2B) and acrylamide gel disk electrophoresis runs on HN2-treated (1 mg/ml HN2, 0.9% saline, 8 hr at 37°) and control hyaluronate were obtained. The intrinsic viscosity was reduced by 10 to 50 per per and a typical gel filtration elution pattern showed retardation by about 10 per cent after HN2 treatment. No gross difference in disk electrophoretic pattern was noted. Similar intrinsic viscosity changes were observed after HN2 treatment of rheumatoid synovial fluids.

X-Rays and Nitrogen Mustard: Independent Action in Chinese Hamster Cells

We have studied the combined effects of X-irradiation and nitrogen mustard treatment on the colony-forming ability of Chinese hamster cells. In contrast to X-irradiation, nitrogen mustard acting by itself yields an exponential dose-effect relationship. Moreover, whether delivered in immediate sequence or after varying intervals, nitrogen mustard treatment does not affect radiation survival and vice versa. These results, plus the lack of any indication of the repair of sublethal nitrogen mustard damage, are consistent with independent but different modes of action at the same site(s), or action at different sites.

The persistence of parental genome segregation in Penicillium chrysogenum after nitrogen mustard treatment

The persistence of parental genome segregation in Penicillium chrysogenum after nitrogen mustard treatment

Fertility-deficient E. coli K-12 mutants.

Fertility-deficient and f2-r mutants of E. coli K-12 were studied. The above mutants were isolated following the nitrogen-mustard treatment of the E. coli K-12 Hfr and E. coli F 1 lac +-strains. Isolation of these mutants from F 1-strains showed that mutations occur in the F-factor no matter whether it was in autonomous state or integrated in a chromosome. The existence of mutants of two types, fertility-deficient and f2-r, was demonstrated. Type I mutants were characterized by the maintenance of a low level phage f2-adsorption activity and by a 10-fold decrease of their fertility as compared with the original strain when crossed in the liquid medium. In crosses in solid media the recombination frequency in the case of type I mutants used as donor was the same as with the original strain. Type II mutants were characterized by the entrie loss of their f2-phage adsorption ability, by a 1,000-fold decrease of fertility in liquid media, and by the inability to recombinate in solid media.

The biology of large solid tumours regressing with nitrogen mustard treatment: a study of the mouse plasma cell tumour Adj-pc-5 and the Walker carcinosarcoma 256.

The biology of large solid tumours regressing with nitrogen mustard treatment: a study of the mouse plasma cell tumour Adj-pc-5 and the Walker carcinosarcoma 256.

Inter-strand crosslinking of DNA by nitrogen mustard

The effect of reaction with nitrogen mustard on the denaturability of DNA was studied in order to clarify the nature of the effect that has been attributed to inter-strand crosslinking. Reaction of bacterial DNA with concentrations of nitrogen mustard as low as 5 × 10 −7 M , corresponding to alkylation of approximately 0·005% of the bases, converted an appreciable fraction of the DNA to a form that did not denature after exposure to dilute sodium hydroxide or to formamide. The hydrogen-bonded structure of the DNA bihelix is broken down by such exposures, but the altered fraction of the DNA reverts to the bihelical form after removal of the denaturant. (Unreaeted DNA remains almost completely denatured after these exposures.) Several physical properties of this recovered bihelical DNA were measured and were found to be similar to those of the original DNA. Measurements of the fraction of the DNA rendered resistant to denaturation were performed by CsCl density-gradient equilibrium ultracentrifugation. Studies with density-hybrid DNA showed that the denaturation resistance produced by nitrogen mustard treatment correlates with an inhibition of strand separation. When DNA was subjected to a mild HN2† treatment and then exposed to alkali at various temperatures, the fraction of the DNA denatured was found to be constant over a wide temperature range. This indicates that the denaturation resistance is mediated by covalent bonds. Denaturation-resistant fractions were produced by the reaction of DNA with the bifunctional nitrogen mustard, HN2, and with bifunctional epoxides. Mono-functional analogues of these agents did not produce this effect. The requirement for sequential reactions of two functional groups was associated with a time-lag in the appearance of denaturation-resistant molecules during reaction with HN2. The formation of denaturation-resistant DNA was initially first-order with respect to HN2 concentration. This indicates that a denaturation-resistant DNA molecule can be produced by the reaction of a single molecule of HN2. The results indicate that nitrogen mustard generates covalent crosslinks between the paired strands of bihelical DNA, and that a single such crosslink per DNA molecule prevents denaturation. Studies with 14 C-labeled HN2 showed that only a small fraction, about 4%, of the DNA-bound mustard molecules became effective crosslinks, under the reaction conditions employed.

Heterozygous diploids of Penicillium chrysogenum and their segregation patterns.

Several heterozygous diploids were made between genetically labelled derivatives of two strains ofPenicillium chrysogenum which produced relatively large amounts of penicillin and were of divergent lineage. The derivatives were labelled with spore colour and nutritional mutations. The diploids, although uniform in having wild type spore colour and being prototrophic, ranged from types having penicillin yields close to that of the original parents to types having less than a quarter of this titre level. Intermediate types had titre levels of about half to threequarters that of the high yielding diploids. Segregants were selected which had arisen naturally and also after nitrogen mustard treatment; most had the spore colour and auxotrophic phenotype of one or other immediate parent. From diploids of low and intermediate titre only haploid segregants with the genetical markers of one parent could be recovered with intact penicillin yield; haploids with the genetic markers of the other showed a marked reduction in yield. However, from diploids of high yield, both parental types could be recovered showing no loss of their original penicillin yield. The bearing of these results is discussed on the suggestion that different degrees of homozygosity between diploids may account for the titre variation observed. An alternative suggestion that mutations suppressive to penicillin titre might cause such variation is also considered.

THE ROLE OF INTERSTITIAL CELLS IN THE MAINTENANCE OF HYDRA. II. BUDDING.

AbstractAnimals whose interstitial cells have been selectively destroyed by nitrogen mustard continue to form new buds for nine days after mustard treatment. Buds present on parent animals at the time of mustard teatment continued to develop at the same rate as control animals; however, in most cases they failed to detach as quickly as control forms.Extensive histological examination revealed a lack of mitoses in gastrodermal digestive cells after nitrogen mustard treatment. Divisions continued in epidermal epithelio‐muscular cells for as long as 20 days. It is suggested that cell divisions in the epidermis are necessary for the initiation of the budding process and that under the present conditions the gastrodermal cells are pulled along passively into the bud outgrowth.

Systemic thiosulfate protection during fractionated regional nitrogen mustard therapy

<h2>Summary</h2> A laboratory study in dogs of the effect of intravenous sodium thiosulfate during fractionated intra-arterial nitrogen mustard treatment revealed partial protection from systemic toxicity. The protective action appeared to be more effective when the thiosulfate was administered simultaneously rather than by pretreatment. The protective doses of sodium thiosulfate used did not appear to reduce significantly the local effect of the nitrogen mustard in treated limbs. However, avoidance of systemic toxicity without significant reduction in observed local effect was also achieved without thiosulfate simply by a minimal reduction of the total dose of HN2 administered fractionally. Similar studies in man with equivalent doses of sodium thiosulfate failed to reveal protection from systemic effects of intra-arterial nitrogen mustard. Since inactivation by sodium thiosulfate in vitro is accomplished by a reaction with the active intermediary metabolite of nitrogen mustard (chlorimine mustard), this agent was also employed in a clinical thiosulfate study, and good protection from systemic toxicity was achieved. Alteration of tumor response in man by the addition of sodium thiosulfate to the treatment could not be completely evaluated by these studies. It is concluded that sodium thiosulfate is of little practical value during fractionated intra-arterial nitrogen mustard therapy, but the use of this protective agent during regional therapy with the active intermediate (chlorimine mustard) is worthy of further clinical trial.

&lt;I&gt;Aspergillus niger&lt;/I&gt;のγ線照射による人工変異

In continuance of earlier studies concerned with nitrogen mustard treatment and uv irradiation, an investigation was carried out to obtaln high citric acid-yielding autants of A. niger by Co/sup 60/ gamma irradintion. The spore suspensions were exposed to various doses ranging from 1.75 x 10/sup 4/ to 21.0 x 10/sup 4/ r, and survival rate and mutation rate were measured. When each sample was subjected to irradiation with 10 x 10/sup 4/ r or more, the survival rate was less than 4% and the mutation rate about 17%. The mutants obtained were classified into seven types by morphologic characteristics. The proportion of markedly or completely repressed sporulation types to all mutants increased as compared with those after nitrogen mustard treatment and uv irradiation. The mutant G-75, classified as sterile type (completely repressed sporulation), produced citric acid in a yield of 55% without the addition of methanol to the glucose-salts medium. When the mutant G-73, classified as normal type (the same as the parent strain in morphological appearance), was used, the addition of methanol greatly promoted the production of citric acid and the yield of citric acid was 71% of the sugar added to the medium. (BBB)

Intestinal vascular permeability changes induced by radiation or nitrogen mustard.

The appearance of an I131-labeled plasma protein substitute, polyvinylpyrrolidone (PVP), in the gastrointestinal tract was increased following X-irradiation or nitrogen mustard treatment of rats. Previous work suggests that leakage of PVP into the intestine may be used as an index of capillary permeability to large molecules. On this basis radiation and nitrogen mustard appear to act by a common mechanism on vascular permeability, as indicated by the similarity in the magnitude and duration of response to them. Partial-body irradiation showed that the upper small intestine was probably the principle site of leakage, and that the effect was caused by direct action on the intestinal epithelium. It was not caused by an increased excretion in the bile nor by an accumulation of secretions which were not reabsorbed because of impaired mucosal function. Treatment of X-irradiated animals with hesperidin-ascorbic acid, diisopropylfluorophosphate, or serotonin had no effect on PVP excretion but prophylactic administration of cysteine did decrease leakage.

Some aspects of myogenesis in vitro.

Suspensions of embryonic chick leg muscle cells have been employed to establish replicate monolayer cultures. Such cultures grow rapidly to form a confluent layer of cells. Despite culture conditions generally assumed to be "dedifferentiative," a high degree of differentiation is attained in terms of the development of cross-striated myofibrils and contractility. To evaluate the possible role of in situ nuclear replication in the development of multinuclearity in muscle cells, an inhibitor of deoxyribonucleic acid (DNA) synthesis, methyl-bis (betachloroethylamine) (nitrogen mustard), was employed. Treatment with nitrogen mustard at concentration levels that profoundly inhibit DNA synthesis does not block the formation of multinuclear cells. On the basis of the pattern of nuclear enlargement after nitrogen mustard treatment, the cytologic picture of treated cultures is interpreted as indicating that the nuclei of only mononucleated cells are normally capable of proliferation. An absence of proliferative activity in the nuclei of multinuclear cell suggests that myoblast proliferation is self-limiting in this system and may explain, in part, the high degree of differentiation attained in monolayer culture.

THE DISSOCIABILITY OF DEOXYRIBONUCLEIC ACID SYNTHESIS FROM THE DEVELOPMENT OF MULTINUCLEARITY OF MUSCLE CELLS IN CULTURE

The effects of a nitrogen mustard on both the morphology and several synthetic capacities of embryonic chick skeletal muscle cells in monolayer culture have been examined. Concentrations of nitrogen mustard which profoundly inhibit deoxyribonucleic acid synthesis specifically do not inhibit the development of multinuclearity in the contractile "ribbons" which form rapidly in culture. Nitrogen mustard affects the nuclear morphology of "fibroblast-like" and multinuclear muscle cells differentially. The mononucleated cells in treated cultures exhibit extreme nuclear enlargement which distinguishes them from the multinuclear cells as well as from both types of cells in control cultures. The nuclei of the multinuclear cells which form after nitrogen mustard treatment, however, do give evidence of having been affected by the treatment. They exhibit somewhat less uniformity of size than similar cells in control cultures. Analogous differences were described by Bodenstein (3) between potentially proliferating cells and postmitotic differentiating cells, marked nuclear enlargement being characteristic of cells in the proliferative zone. The results are more compatible with the hypothesis that multinuclearity arises through successive cell fusion than through amitotic nuclear multiplication, since it is unlikely that any form of nuclear replication could occur in the absence of DNA synthesis.

Depression on Normal Serum Bactericidal Activity by Nitrogen Mustard

Summary Single intravenous injections of 2.5, 3.0 or 3.5 mg of nitrogen mustard/kg caused a depression in the bactericidal activity of rabbit serum against Bacillus subtilis. The loss of serum bactericidal activity did not take place until about the third post-treatment day and returned to normal between the 14th and 20th days after nitrogen mustard administration. An intravenous injection of L-cysteine immediately prior to the administration of nitrogen mustard decreased the length of time that serum bactericidal activity was depressed following nitrogen mustard treatment. Nitrogen mustard treatment caused no statistically significant difference in either the natural agglutinins against Bacillus subtilis or circulating complement. Both circulating leukocytes and serum properdin were depressed by doses of nitrogen mustard that suppressed serum bactericidal activity. The serologic alterations, leukocytic changes, and the depression of serum bactericidal activity which follows near-lethal doses of nitrogen mustard were almost identical to those that follow exposure to near-lethal doses of total body x-irradiation.

Studies with mustards. IV. Effects of nitrogen mustard treatment of deoxyribonuclease and deoxyribonucleic acid upon the enzymic degradation of deoxyribonucleic acid

Treatment of either deoxyribonuclease or deoxyribonucleic acid with nitrogen mustard caused a decrease in the rate of enzymic degradation of deoxyribonucleic acid, and the treatment of the nucleic acid caused a greater decrease than treatment of the enzyme. The significance of this finding is discussed.

Reversional mutations in the methionine-requiring strain of &lt;i&gt;Ustilago maydis&lt;/i&gt;

The frequency of reversions, of the methionine-requiring mutant strain (4-24) of Ustilago maydis occurred spontaneously as well as by the X-ray irradiation and tris-nitrogen mustard treatment was studied and the nature of the reversions was discussed.1. Spontaneous reversion takes place around at the rate of 3.0×10-7 per cell per division, when cultured in the complete liquid medium.The rate of reversional mutation was increased by both X-ray irradiation and nitrogen mustard treatment up to the orders of 10-4. The mutation constant is calculated to be 2.2×10-7 per roentgen.2. Three types of reversions are discerned:The pseudo reversion type (PR) appears as a result of residual growth and syntrophism. The complete reversion type (CR) and the inferior reversion type (IR) appear as a result of one gene mutation. The IR-type is distinguished from the CR-type by the restricted growth during early subcultures.

Nitrogen mustard as an adjunct to radiation in the management of bronchogenic cancer.

It is generally agreed that pulmonary resection offers the patient with bronchogenic carcinoma his best chance for cure. Unfortunately, most cases are inoperable when first seen. Of the 400 patients with bronchogenic carcinoma seen at the Veterans Administration Hospital in the Bronx (N. Y.) in the past four years, 90 per cent came too late for surgery. At the Hines Veterans Hospital, 95.4 per cent of 1,057 such patients admitted from 1937 to 1947 were already beyond any attempt at salvage by surgical resection (1). This distressing picture is reflected in other hospitals and clinics. It may be estimated that in 1949 there were in this countrya bout 28,000 new patients with bronchial cancer, compared to 10,000 in 1939 (34). Unless treated by irradiation, inoperable bronchogenic carcinoma runs its lethal course rapidly and inexorably, complicated by distressing signs and symptoms, both local and systemic. Of 584 non-irradiated inoperable patients, not one survived more than one year after the onset of the disease, according to the combined reports of Leddy and Moersch (18) at the Mayo Clinic, Widmann (31) at the Pennsylvania General Hospital, and Ariel et al. (1) at the Hines Veterans Hospital. On the other hand, our own experience with the roentgen treatment of 605 inoperable cases in the last fifteen years has demonstrated that, when radiation therapy was feasible, effective relief from intractable local symptoms was often possible. Indeed, prolongation of useful life for many months, and even years, was sometimes accomplished, particularly when the patient's physical condition was such that cancerocidal doses (5,000 to 6,000 r) could be approached. An occasional rare cure may be achieved. These observations are supported by the experience of others in a group of 508 roentgen-treated cases reported in the literature (6, 18, 28, 31). It is not possible, however, to give to every inoperable patient the benefits which irradiation can offer. At intervals in the course of the disease irradiation may seem to be ineffective or, for various reasons, even unfeasible. An effective adjunct to radiotherapy would therefore, appear, to be most desirable during these periods. The object of this paper is to present the results of our investigation of nitrogen mustard or HN2,—methyl-bis (beta chloroethyl) amine hydrochloride—employedin this role.4 Rationale of Nitrogen Mustard Treatment The early investigations of Gilman (11), Goodman (12), Jacobson (13), and Karnofsky (14 and 15), and their co-workers, with nitrogen mustard in human lymphomas, excited our interest in investigating its role as an adjunct to radiotherapy in a variety of malignant disorders. The study was begun in the Radiation Clinic in January 1947, in collaboration with the Committee on Growth of the National Research Council. Bronchogenic carcinomas were included because of scattered reports of clinical benefit by other investigators (33).

CHEMICAL MUTAGENESIS

CHEMICAL MUTAGENESIS

CLINICAL EXPERIENCE WITH NITROGEN MUSTARD THERAPY

RECENT reports of results obtained with the use of nitrogen mustard derivatives have aroused much speculation regarding the efficacy of chemotherapy in certain malignant neoplastic diseases. The Veterans Administration Hospital, West Los Angeles, Calif., has been designated as a center for the treatment of tumors. This provided a relatively large number of patients who had leukemias and lymphomas, and it was decided to evaluate the effects of nitrogen mustard therapy on a suitable group. METHOD AND RESULTS Method. —The routine study included a search for enlarged lymph nodes, examination of the liver and spleen, complete blood count, sternal puncture, roentgenologic study of the chest and biopsy. The patient was then presented to the members of a Tumor Board, who selected the type of therapy to be used in each instance. In those patients for whom the nitrogen mustard treatment was recommended, the bis form of the drug was administered intravenously,