Oral administration of BCG as an adjuvant to surgical treatment of carcinoma of the bronchus. A controlled clinical trial of the value of bacille Calmette--Guérin (BCG) vaccine given orally to patients with resectable carcinoma of the lung was conducted in 18 centres across Canada. A total of 308 patients were included in the analysis, 155 in the BCG group and 153 in the control group. The two groups were similar at the time of admission to the trial. BCG (120 mg) was given orally at weekly intervals for 1 month, every 2 weeks up to 3 months and then every 3 months until the total duration of therapy was 18 months. Over a 3- to 5-year follow-up period after the operation there was no difference in survival between the two groups, the proportion alive at 2 years being 61% in the BCG group and 58% in the control group. There was also no evidence of differences in the time to the detection of recurrent or metastatic disease or in the distribution of such disease. An analysis of prognostic factors confirmed the poor survival associated with histologically confirmed lymph node involvement. It may be concluded that no favourable effect from the oral administration of BCG was demonstrated.


Dietary fibre and colonic neoplasia.

Dietary fibre and colonic neoplasia. Dietary plant fibre, or plantix, is thought to play a significant role in the pathogenesis of colon cancer in humans. It is a complex polymeric substance that has several distinct components resistant to hydrolysis by the digestive enzymes of humans. These components include cellulose, hemicelluloses, pectins, lignin, gums, mucilages and, in certain instances, algal polysaccharides. These polymers have different physicochemical properties, and recent evidence from experimental studies in animals treated with carcinogens suggests that some may exert protective effects in the intestine and others may enhance colon carcinogenesis. This review synthesizes information on the chemical composition, methods of analysis and physicochemical properties of dietary plant fibre and reviews available studies examining the role of fibre in colonic neoplasia in animals and humans.


Report of a clinical trial of a Canadian preparation of antihemophilic factor. A clinical trial of a Canadian preparation of antihemophilic factor has been completed. The mean percentage increase in the factor VIII coagulant activity in the plasma was determined to be 2.1 +/- 0.8 per unit per kilogram dose, which compares very favourably with published data. The mean half-life of antihemophilic factor was found to be 14.7 hours. Side effects and reactions to the product were minimal and the product has been rated as clinically effective. No significant differences between lots were noted in the in vivo response or the half-life.


Familial hypogonadotropic hypogonadism with alopecia. In one family several male and female members had hypogonadism and frontoparietal alopecia, whereas other members with normal sexual development had normal scalp hair. Clinical and laboratory evaluation of three affected young men (two brothers and their cousin) revealed that the hypogonadism was the result of decreased serum concentrations of follicle stimulating and luteinizing hormones. There was no evidence of a deficiency of any other pituitary hormone. Long-term treatment of the three patients with human chorionic gonadotropin resulted in an increase in the serum testosterone concentration, the appearance of male secondary sex characteristics and an increase in the size of the external genitalia.


The Canadian certification examination in psychiatry. II: Who passes and who fails. No analysis of Canadian certification examinations in psychiatry has previously been published although analyses of the American Board of Psychiatry and Neurology and the British Membership examinations are available. Because candidates, directors of residency training, mental health planners and consumers are all interested in who passes and who fails the certification examinations, available examination data for English speaking candidates are analyzed. Successful candidates are more likely to be younger; to have attended medical schools in English speaking countries (in North America, the British Isles, or the "old Dominions"); to have placed in the upper two-thirds of their medical school class; to have entered psychiatric training soon after graduating from medical school and then to have completed their training without interruption. Some limitations of the examinations and the problem of candidates who fail are briefly discussed.


The Canadian certification examination in psychiatry. III. Towards better certification techniques. Although there has been much criticism of specialty certification examinations there is general agreement that they are an important safeguard of competence in medical specialties where the consumer cannot judge this. The Canadian Royal College has made strenuous attempts to improve certification techniques but these have not been heretofore widely reported. This paper reports on the rationale for the replacement of the essay examination by multiple choice questionnaires, the problems associated with MCQ, and the efforts made to improve the fairness, validity and reliability of the clinical (oral) examination. These efforts have been in the areas of selection of examiners, training for examiners, standardization of marking and the conduct of the examination. A description is given of the conduct of the examination and what the examiners look for in the candidate's performance. The development and increasing importance of the In-Training Evaluation are discussed. The publication of A Resident's Guide to Psychiatric Education with multinational participation advances the possibility of future reciprocity in psychiatric examinations conducted in several English speaking countries.


The relationship between agoraphobia and primary affective disorders. Detailed clinical records were kept on a series of 55 agoraphobic patients who presented to a general hospital psychiatric practice over a 3 year period. A review of the records revealed that 91% of these patients were diagnosed as suffering from a unipolar or bipolar primary affective disorder. The anxiety and phobic symptoms tended to mask the presence of the affective disorder. This observation is consistent with most of the published data on the agoraphobic syndrome. It could also explain the inconsistent effects of treatment of agoraphobia compared with simple phobias. The possible biological and psychological connections between primary affective disorders and the agoraphobic syndrome are discussed.


The limits of Munchausen's syndrome.

The limits of Munchausen's syndrome. The authors review the literature on Munchausen's syndrome and speculate about possible underlying psychological mechanisms. The proposed DSM III classification of factitious illnesses suggests a continuum from hysteria on one end of the spectrum to malingering on the other. Two case studies are presented which represent variants of this syndrome. Both patients were given a sodium amytal interview, a procedure not previously reported in the Munchausen's literature. The procedure was helpful in eliciting a more accurate history and a clearer sense of the underlying dynamics. Some suggestions for further research are made.


Civil commitment practices in 1977: troubled semantics and/or troubled psychiatry. This paper examines the practice of involuntary mental hospitalization through examination of criteria used for committment in a sample of 200 civil commitment certificates. Special reference is made, following previous research, to criteria used to describe the person deemed dangerous to himself or to others. The relevance of the findings of present practices in mental health is discussed. New procedures which facilitate presentation of factual evidence, and which eliminate gratuitous information, are required without delay.


Length of hospitalization predicted by self assessment of social competence. The purpose of the study was to assess the feasibility of using demographic and social competence information to predict length of stay in a psychiatric hospital. The results indicated that social competence variables were much better predictors of length of stay than demographic variables. Moreover, no combination of demographic and social competence variables led to more accurate predictors than social competence information alone.


The psychoanalytic theory of depression. Part I--The major contributors. This article is the first of a two part review of the psychoanalytic theory of depression. In this first part of the review the work of the major contributors to the psychoanalytic theory of depression is discussed. The writings of Abraham, Freud, Rado, Klein, Jacobson, Benedek, Bibring, Spitz, Sandler and Bowlby, among other, are presented and critically reviewed by the author. The work of these authors has been selected for this review because they have made the most seminal contributions to the development of the psychoanalytic theory of depression. Necessarily those authors whose contributions have been largely clinical have not been included, the major focus of this review being theoretical. In reviewing the writings of the major contributions the major themes in the theory of depression can be discussed. These themes will form the subject of the next paper in this review.


Induced parturition in swine herds.

Induced parturition in swine herds. Intramuscular injections of various levels of prostaglandin F(2alpha) were administered to 116 pregnant swine in three commercial herds and the University research unit on Day 111, 112 or 113 of gestation. Sixty-three percent of the treated animals farrowed during the working day immediately following treatment. The parturition time, birth weight, litter size, number of piglets weaned, growth of the piglets to weaning and subsequent rebreeding of the dams were comparable with the expected performance in the herds. The results indicated that prostaglandin F(2alpha) in doses ranging from 5 to 12.5 mg per animal were effective for induction of parturition in a substantial proportion of the treated animals. Successful induction of parturition could reduce the farrowing interval for batches, allow more supervision of farrowing, facilitate transfer of piglets from large to small litters and generally increase efficiency in the farrowing unit. This technique could have practical application in intensive swine production units.


Torsion of the uterus--a cause of colic in the mare. A case of uterine torsion in a mare with colic is described in which an early diagnosis was made and the torsion successfully reduced through a standing laparotomy with survival of both the mare and the foal. This case is used to stress the importance of thorough examination of all pregnant mares with colic in order to differentiate uterine torsion from other causes of abdominal pain, thereby avoiding delay in surgical correction and reducing the risk of fetal and/or maternal death. The advantages of the standing laparotomy are presented in support of this method of treatment of torsion of the gravid uterus.


Amelioration of adriamycin skin necrosis: an experimental study. Skin necrosis following subcutaneous Adriamycin extravasation is a significant clinical problem. In this study Adriamycin dilution and various drugs were tested for their ability to ameliorate this toxic effect. When Adriamycin was diluted to a concentration of less than or equal to 0.25 mg/ml, no skin ulceration was observed. Drugs such as hydrocortisone, dexamethasone, lidocaine, bupivacaine, phentolamine, and hyaluronidase were ineffective in modifying Adriamycin skin necrosis. Care in iv drug administration and dilution of Adriamycin are the best means of preventing the sequelae of subcutaneous Adriamycin extravasation.


Influence of L-tyrosine phenol-lyase on the growth and metabolism of B16 melanoma. Administration of L-tyrosine phenol-lyase inhibited growth of established B16 melanomas in BDF1 mice. Under the conditions used, the enzyme exerted a cytostatic effect on the tumors. The addition of L-tyrosine phenol-lyase to growing melanoma cultures blocked cell division and caused an accumulation of cells in the G1 (G0) pultures was suppressed, the most pronounced effect of L-tyrosine phenol-lyase was a rapid and near-complete cessation of uridine uptake and incorporation. This block in RNA synthesis was also observed in human fibroblasts treated with L-tyrosine phenol-lyase.


Biochemical properties and immunogenicity of L-phenylalanine ammonia-lyase: effects on tumor-bearing mice. L-Phenylalanine ammonia-lyase (PAL) from yeast was used to deplete plasma L-phenylalanine and L-tyrosine in an attempt to achieve inhibition of tumor growth in mice. Plasma L-phenylalanine and L-tyrosine were reduced to nondetectable levels when circulating PAL activity was maintained at greater than or equal to 0.06 unit/ml. Repeated administration resulted in the appearance of anti-PAL antibodies. A radioimmunoassay based on the method of Farr was developed to determine quantitatively the presence of anti-PAL. Sublethal total-body irradiation temporarily suppressed the immunologic response of the host. Long-term specific immunosuppression to PAL was achieved with cyclophosphamide (CPA). A single dose of 180 mg/kg of CPA administered ip to mice 24 hours before, simultaneously with, or 24 hours after 100 units/kg of PAL induced tolerance for 450 days (20 injections of enzyme). The plasma half-life of PAL in CPA-treated mice remained essentially the same as that found after a single injection (25 hours), and anti-PAL probably will require specific immunosuppression of the host to repeated injections of the enzyme.


Tumor therapy by deprivation of L-methionine: rationale and results. Published reports indicate that normal rodent cells can grow in medium containing either L-methionine or L-homocysteine, whereas malignant rodent cells have an absolute requirement for L-methionine. Our studies with two normal human cell lines (fetal lung fibroblasts and bladder epithelial cells) exhibit equal growth in media containing either L-methionine or L-homocysteine. The same is true for five malignant human cell lines (carcinoma of the cervix [HeLa], adenocarcinoma of the breast [AlAb], acute lymphoblastic leukemia [MOLT-3], Wilms' tumor [SK-NEP-1], and reticulum cell sarcoma [T-77], whereas four other malignant cell lines (adenocarcinoma of the breast [SK-BR-2-III], the two lymphoblastic leukemias [CCRF-HSB-2 and CCRF-SB], and a neuroblastoma [SK-N-MC]) have absolute requirements for L-methionine. Two malignant cell lines, an adenocarcinoma of the lung (A549) and an adenocarcinoma of the pancreas (Capan-1), showed restricted growth under the experimental conditions used. L-Methionlinase (L-methionine-alpha-deamino-gamma-mercaptomethane-lyase, EC 4.4.1.11) at a concentration of 0.1 unit/ml leads to complete growth inhibition of cell cultures of both the normal human fetal lung fibroblasts (F-136-35-56) and the acute lymphoblastic leukemia (CCRF-HSB-2). L-Homocysteine-thiolactone in medium containing L-methioninase could partly "rescue" the normal but not the malignant cells.


L-cyst(e)ine requirements of malignant cells and progress toward depletion therapy. The L-cyst(e)ine requirements of normal and malignant cells are reviewed and expanded within the context of establishing whether the measurement of gamma-cystathionase levels constitutes a predictive test for tumor sensitivity to L-cyst(e)ine depletion. The ability of both purified L-cysteine desulfhydrase and gamma-cystathionase to inhibit the growth of the L-cystine-dependent L1210 leukemia in culture is presented, as well as approaches to circumvent the limitations of these enzymes for in vivo therapy. The ability of proparagylglycine to inhibit L-cysteine biosynthesis in vivo is reviewed for its possible use in combination therapy. In addition, the ability of poly D,L-alanine modification of Escherichia coli L-asparaginase to increase the plasma half-life in mice tenfold as well as to decrease the immunogenicity of the enzyme is presented.


L-Threonine deaminase as a possible antitumor agent. In vitro studies with L-threonine deaminase purified from sheep liver demonstrated that the enzyme was cytotoxic toward two lines of murine leukemia cells but not toward L cells or normal human fibroblasts. When L-threonine deaminase was injected into mice, it was possible to lower the plasma L-threonine concentration by 90%. The enzyme had a half-life of 1.5 hours in the circulation.


Enhanced effect of an L-glutamine antagonist, L-(alphaS,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid, by Acinetobacter L-glutaminase-L-asparaginase. The effect of L-glutamine and L-asparagine depletion by Acinetobacter L-glutaminase-L-asparaginase on the toxicity and antitumor activity of L-(alphaS,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (NSC-163501) was tested in mice. The LD50 of six daily doses of NSC-163501 in BDF1 female mice decreased from 7.5 to 0.3 mg/kg/day by combination treatment with the enzyme. Enzyme therapy also decreased the dose of NSC-163501 needed for maximal prolongation of survival in these mice inoculated with L1210 leukemia. Nevertheless, the combination did not prolong survival in L1210-bearing mice beyond that of higher doses of NSC-163501 alone. In contrast, the combination of enzyme plus NSC-163501 inhibited the growth of established sc implanted Ehrlich ascites carcinoma in ICRf male mice much more than either agent alone. Treatment with Acinetobacter L-glutaminase-L-asparaginase decreased the L-asparagine and L-glutamine levels in acid extracts of the Ehrlich tumor. NSC-163501 did not affect the amide levels or alter the decrease produced by enzyme therapy.


Treatment of L5178Y tumor-bearing BDF1 mice with a nonimmunogenic L-glutaminase-L-asparaginase. An L-glutaminase-L-asparaginase from Achromobacter has been rendered nonimmunogenic by the covalent attachment of polyethylene glycol (PEG) to nonessential amine groups of the enzyme. PEG-L-glutaminase-L-asparaginase exhibits a greatly enhanced half-life in the bloodstream compared to the unmodified enzyme in normal mice, and is effective in prolonging the survival of BDF1 mice inoculated ip with L5178Y cells. PEG-L-glutaminase-L-asparaginase appears rapidly in the blood following ip injection.


In vitro system for detection of antimetabolites of specific amino acids. An in vitro system was developed to detect antimetabolites in fermentation liquors of soil microorganisms. This system effectively uncovers antimetabolites of purines and pyrimidines and of selected amino acids with established differences in their biosynthesis by normal cells versus certain malignant cells. Currently these amino acids include: L-asparagine, L-aspartic acid, L-glutamine, L-cysteine (cystine), L-methionine, L-arginine, L-histidine, L-tyrosine, L-phenylalanine, L-tryptophan, L-threonine, and L-serine. It is expected that these antimetabolites could be useful either alone or in combination with specific depleting enzymes in the treatment of malignancies where such an imbalance was established.


Effect of a forced diuresis on the distribution and excretion (via urine and bile) of 195mplatinum when given as 195mplatinum cis-dichlorodiammineplatinum(II). 195mPlatinum cis-dichlorodiammineplatinum(II) (195mPt cis-DDP) was injected iv with furosemide or mannitol into male Sprague-Dawley rats. The effect of such a diuresis on the distribution, excretion, and retention of cis-DDP was measured. The mannitol-treated animals had a significant increase in the urinary excretion of 196mPt over control animals for the first five 15-minute collection times and for the 24-hour cumulative urine for the 0.01-mg/kg dose. At the 6-mg/kg dose, only the first three 15-minute and 24-hour cumulative urine collections were significant. This increased excretion, however, did not affect tissue or blood concentrations of this agent. Furosemide did not affect the excretion of 195mPt. A biliary excretion of 195mPt given as 195mPt cis-DDP is documented. The excretion is biphasic, with a rapid phase followed by a slower protracted phase. Forced diuresis, if it affords renal protection, does so during the first 90 minutes of urinary excretion.


Biologic activity of MCNU: a new antitumor agent. A novel nitrosourea derivative, methyl-6-[[[(2-chloroethyl)nitrosoamino]carbonyl]-amino]-6-deoxy-alpha-D-glucopyranoside (MCNU), is a water-soluble compound in which a methoxyl group is attached to the C-1 position and an N-(2-chloroethyl)-N-nitrosoureido group is attached to the C-6 position of the glucose moiety. MCNU exhibited a marked life-prolongation or growth-inhibitory effect against mouse L1210 leukemia, adenocarcinoma 755, Nakahara-Fukuoka sarcoma, Lewis lung carcinoma, and B16 melanoma. Ip, oral, or iv administration of MCNU was markedly effective against L1210 leukemia, and the therapeutic ratio by ip administration was larger than that of chlorozotocin or CCNU. The life-prolongation effect of MCNU against established Lewis lung carcinoma was similar to that of methyl-CCNU. The bone marrow toxicity of MCNU was less than that of CCNU but considerably more than that of chlorozotocin.


Levodopa and dopamine analogs: melanin precursors as antitumor agents in experimental human and murine leukemia. L-Dopa methyl ester has been shown to be a novel antitumor agent. Furthermore, the L-dopa analogs, D-dopa, alpha-methyldopa, and dopamine, also exhibit significant antitumor activity in the L1210 and P388 lymphocytic leukemia systems. Structure-activity studies confirmed that the presence of a catechol moiety was essential for activity. Two analogs, 3,4-dihydroxybenzylamine and N-acetyldopamine, which were much less neurotoxic, exhibited the greatest antitumor activity. In vitro, at concentrations from 0.5 to 3.0 mM, there is a rapid and profound inhibition of radiolabeled thymidine incorporation as compared to uridine or leucine incorporation. Continuous exposure of exponentor up to 24 hours resulted in a block of traverse of cells through the cell cycle in G1 coupled with a depletion of cells with a G2 complement of DNA. In vivo, toxicity of these compounds appears to be mediated principally by conversion to dopamine. Similar effects upon thymidine incorporation were observed in human leukemia cells in vitro.


An effective conscious animal model of atrial fibrillation. In 11 fully conscious dogs with chronically implanted spicardial electrodes, 50 Hz sine-wave electrical stimulation of the left atrium reliably and repeatedly produced an arrhythmia which was indistinguishable from atrial fibrillation in terms of its ECG appearance, the statistical properties of the ventricular response and responses to a number of pharmacological agents. In five of the 11 preparations this arrhythmia consistently persisted for periods in excess of 10 min following the cessation of stimulation, indicating an intrinsic cardiac basis for the arrhythmia and suggesting that it is true atrial fibrillation. A close correlation between the ventricular response to this arrhythmia and the degree of atrioventricular conductivity, together with the observation that dramatic changes can occur in ventricular responses without corresponding changes in atrial activity, suggest that the ventricular response is mainly a function of the atrio-ventricular conducting system. The experimental model described is useful for the study of both short and long term drug action no atrial fibrillation and for the evaluation of methods used therapeutically in attempts to terminate episodes of paroxysmal atrial fibrillation.


Opposing effects of deoxycorticosterone and spironolactone on isoprenaline-induced myocardial infarction. Adult, male Sprague-Dawley rats were subjected to massive myocardial infarction with isoprenaline. Some of the animals were treated with the potent mineralocorticoid, deoxycorticosterone, or the anti-mineralocorticoid, spironolactone. Autopsies spaced at sequential time intervals demonstrated that circulating levels of enzymes, eg, CPK, SGOT, SGPT and LDH, lipids, eg, triglycerides, free fatty acids, and cholesterol, glucose, Bun, and corticosterone rose in conjunction with on-going necrosis and became lowered in conjunction with myocardial repair. Body and organ weights, ie, adrenal, thymus, heart, and kidney, underwent dynamic changes commensurate with necrosis and repair. Animals given isoprenaline alone died (52% survival) during the height of their massive infarct and congestive heart failure (Days 3 and 4). Animals treated with DOC showed the most severe congestion but died (36% survival) during the late repair phase (Days 7 and 8). Animals treated with spironoloactone showed the most effective clearance of thoracic fluid and also dies (18% survival) during the late repair phase (Days 7 and 8). These spironolactone-treated animals had large, left ventricular aneurysms with organised, adherent thrombi. It is believed that alterations in electrolyte balance induced by DOC or spironolactone caused changes in adrenocortical function which greatly modified the usual pathophysiological response to myocardial infarction.


Temporal changes in endocardial energy metabolism following propranolol and the metabolic basis for protection against isoprenaline cardiotoxicity. Propranolol infusions in open-chested, anesthetised dogs increased endocardial levels of ATP and phosphocreatine. It reduced ADP, AMP, glucose-6-phosphate and lactate in this region. Endocardial glycogen was transiently increased. Metabolic perturbations in the endocardium produced by cardiotoxic doses of isoprenaline were prevented or reduced by propranolol pretreatment. Thus, propranolol appears to have a more selective action on endocardial versus midmyocardial or epicardial metabolism.


Resistance of contracting myocardium to swelling with hypoxia and glycolytic blockade. The interrelationship of myocardial metabolism, performance and tissue hydration was examined in isolated contracting rat, guinea pig and dog myocardium. Myocardial metabolism was altered by blocking aerobic, and both aerobic and anaerobic metabolism. Myocardial water content and distribution were measured in rat myocardium using 3H-inulin and 51Cr-EDTA as extracellular markers. Myocardial hydration was also evaluated by light and electron microscopy. The relative susceptibility of non-contracting slices of rat and guinea pig myocardium and kidney to swelling secondary to these interventions was also explored. Hypoxia resulted in a partially reversible reduction in mechanical function; hypoxia plus glycolytic blockade led to irreversible severe contracture and total loss of tension development. Neither hypoxia nor hypoxia plus glycolytic blockade resulted in increased total tissue or extracellular water in previously contracting preparations or in non-contracting slices of myocardium. On the other hand, there were significant increases in cellular water in similarly treated kidney slices after each intervention. Thus, despite severe, irreversible derangements of mechanical function, myocardium did not swell under conditions which produced swelling in renal cortex.


A system oriented electrocardiographic amplifier. A low noise, programmable gain amplifier has been developed for acquiring ECG data from a variety of recording environments. Features include: low noise (4muV peak-to-peak) differential inputs; digitally programmable gains (range 100 to 16000) and output DC offsets (range -3.5 to +4 V) controlled via a serial interface; externally selectable low frequency response (0.04 or 0.08 Hz); and an on-board output monitor multiplexer controlled via a second serial interface.


Morphological characteristics of the spermatogonial stem cells in man. The present investigation is concerned with establishing morphological criteria of spermatogonial stem cells in man. Testicular biopsies from patients having undergone semicastration for malignant tumors and radio- and chemotherapy for one year following the operation were studied light and electron microscopically. Those spermatogonial types that survived the treatment were regarded as stem cells in view of the fact that the stem cells, in contrast to the more differentiated spermatogonia, are radiation resistant and less sensitive to various noxious agents. In 7 out of 28 cases examined, a small number of spermatogonia was found adjacent to the basement membrane. The majority of these cells show the characteristic features of pale type A spermatogonia, while a few cells may represent variants of this cell type. The dark type A spermatogonia are almost completely eliminated from the seminiferous tubules. A concept is proposed that the stem cells of the human testis may be derived from the pale type A spermatogonia or the variants of this cell type.


Ultrastructure of the corpus cardiacum and corpus allatum of the house cricket Acheta domesticus. The ultrastructure of the corpus cardiacum (CC) and corpus allatum (CA) of the house cricket, Acheta domesticus, is described. Axon profiles within the CC contain neurosecretory granules 160-350 nm in diameter which are indistinguishable from those found in type I neurosecretory cells of the pars intercerebralis and in the nervus corporis cardiaci I. The CC itself contains two cell types: intrinsic neurosecretory cells and glial cells. Intrinsic NSC cytoplasm contains Golgi bodies and electron dense neurosecretory granules 160-350 nm in diameter. Synaptoid configurations with 20-50 nm diameter electron lucent vesicles were observed within axon profiles of the CC. The structure of the CA is relatively uniform with one cell type predominating. Typical CA cells possess large nucleoli, active Golgi complexes, numerous mitochondria, and occassional microtubules. Groups of dark staining cells scattered throughout the CA of some animals were interpreted as evidence of cellular death.


Long term increase of mucosal mast cells in the rat induced by administration of compound 48/80. Administration of Compound 48/80 to rats for 5 days resulted in an increase of the specific type of mucosal mast cell, while connective tissue mast cells elsewhere were almost completely degranulated. The number of mucosal mast cells increased slowly for another 5 days and then returned to the control level, in an exponential manner. The half life of the newly formed mast cells was calculated to be about 40 days. This value may be taken as an estimate of the half life of mucosal mast cells. These cells, therefore, constitute a fraction of mast cells with rapid turnover. Available evidence indicates that the classical connective tissue mast cell has a very long life span, without significant turnover in terms of cell death and cell renewal. We suggest that the increase of mucosal mast cells is an indirect effect of Compound 48/80, related to its effect on other mast cells and mediated by material(s) released from these cells during the secretory process.


Ultrastructure and function of the labial nephridia and the rectum of Orchesella cincta (L.) (Collembola). The collembolan Orchesella cincta possesses a well-developed coelomoduct kidney. The presence of podocytes in the wall of the sacculus and the fact that the epithelium of the nephridial tubule has the ultrastructural characteristics of resorbing cells, indicate that this is an "ultrafiltration-reabsorption" kidney. Apparently also the rectum is lined by a reabsorptive epithelium; the cells possess an extensive system of apical and basal infoldings. This view is sustained by the fact that the stereology of the apical channel system varies in animals kept under different moisture conditions. During the intermoult period, both organs are subject to strong morphological changes, which are obviously related to the feeding rhythm.


Morphogenesis of tight junctions in the peritoneal mesothelium of the mouse embryo. The peritoneal mesothelium of mouse embryos (12 to 18 day of gestation) was studied by freeze-fracture and in sections in order to reveal the initial formation of the tight junctions. Freeze-fracture observations showed three types of tight junctions. Type I consists of belt-like meshworks of elevations on the P face and of shallow grooves on the E face. No tight junctional particle can be seen either on the elevations or in the grooves. Type II shows rows of discontinuous particles on the elevations on the P face. Type III consists of strands forming ridges on the P face. On the E face, the grooves of Type II and III appear to be narrower and sharper than those of Type I. Quantitatively, Type I junctions are most numerous during the early stages (day 12-13) of embryonic development, while Type III junctions become more common in the later stages, and are the only type seen by day 18. Observations on sections, however, fail to distinguish between the three types. The results suggest that an initial sign of tight junction formation is close apposition of the two cell membranes in the junctional domain, without tight junctional particles. Later, the particles appear to be incorporated in the tight junctions and the strands form by fusion of the particles.


A study of gonadal organogenesis, and the factors influencing regeneration following surgical castration in Deroceras reticulatum (Pulmonata:Limacidae). At hatching, the hermaphrodite duct of Deroceras reticulatum consists of a single cell type designated the Gonadal Stem Cell (GSC). Proliferation of the GSC leads to the formation of numerous ductules each of which forms one of the acini of the gonad. The germinal and supporting cells are derived entirely from the GSC. The germ cells differentiate first, followed by the Sertoli and follicle cells. At the early sperm stage of gonadal development the hermaphrodite duct differentiates to function as a seminal vesicle. Once the GSC are committed to this change they lose their regenerative ability. The only remaining GSC are the cells of the acinar epithelium, and these retain their germinal potential until the death of the animal. Regeneration will occur from the hermaphrodite duct provided it is in the immature state, i.e., composed of GSC, and is exposed to the hormonal conditions of a young animal. Nervous connections and the presence of an artery are not necessary for this regeneration. The presence of a functional gonad does not inhibit regeneration.


The air-blood barrier in the human lung. A freeze-fracture study. The air-blood barrier was studied in replicas of freeze-fractured lung biopsies collected from healthy human subjects. Adjacent pneumocytes display a belt-like network composed of 3-7 superimposed ridges ("fibrils") on the P face and complementary grooves on the E face, i.e., a structure corresponding to a "tight" junction. On the other hand, adjacent capillary endothelial cells show a continuous system of 2-4 membrane foldings. These appear mainly as smooth surfaced crests on the P face; on the E face furrows are seen, at the bottom of which a row of particles is situated. This arrangement suggests a "leaky" type of junction. Discontinuous occluding junctions are located in the pericytic venular segment of the alveolar vessels. The present findings are in agreement with previous physiological and ultrastructural tracer studies locating the main part of the diffusion barrier for small polar solutes and proteins in the alveolar epithelium. Communicating junctions are demonstrated between type I and type II pneumocytes, indicating intercellular cooperation between these cells of common embryonic origin, but which fulfill different functions in the adult. In the endothelium of the non-muscular alveolar vessels communicating junctions are lacking. Desmosomes occur in the epithelium between type I and type II pneumocytes; square arrays of particles characterize the plasma membrane of type I pneumocytes.


Observations of the pineal region of non-eutherian mammals. A survey has been made of the pineal region of the brain of 11 species of marsupials belonging to 5 families and a species from both families of monotremes. The results show that the pineal body of non-eutherian mammals, although well-defined in all species, has a very varied morphology. Three types of pineal recess occur: (i) a pineal recess in sensu stricto, (ii) an intercommissural pineal recess, and (iii) an infrapineal recess. The existence of nerve fibres which pass through the pineal body and form a spatial link between the habenular and posterior commissures, has been demonstrated in marsupials and monotremes. It is also likely that these animals as well as eutherian mammals possess a nervus conarii. Nerve cells are not a constant feature of the non-eutherian pineal body. The subcommissural organ (SCO) is present in all species. It does not exhibit the same degree of morphological variation as the pineal body. Horizontal sections available for 4 species within 3 families of marsupials show it to be composed of a median portion jointed to bilateral protuberances. Large nerve cells occur within the SCO in all marsupial species; they are absent from the monotreme SCO. Tentatively, the relationship of these neurons to the SCO is considered to be merely one of association. The importance of an extended comparative study of this region in non-eutherian mammals in order to add insight into its phylogeny and function is emphasized.


Quantitation of gastrin and somatostatin cell populations in the antral mucosa of the rat. Comparative distribution and evolution through different life stages. Total antral gastrin and somatostatin cell populations as well as their relative distribution pattern throughout the antrum were studied in rats with advancing age from birth time to old age. Both endocrine cell populations were estimated, after staining by immunoperoxidase technique, with a quantitative method using serial parallel strips from entire stomachs. Gastrin cells were regularly found at less than 1 h of post-natal life, but were few in number (447 +/- 82) cells. Somatostatin cells, not seen at birth, were observed in all rats at seven post-natal days; then they increased in number less rapidly but more regularly than gastrin cells. During the normal adult period, corrected gastrin cell population corresponds to about 330,000-500,000 cells and corrected somatostatin cell populations to about 130,000-200,000 cells. For the whole antrum the ratio of gastrin cell to somatostatin cell populations decreases through the rat life from 6.5 at 7 days to 1.5 in old age with a stable value, 2.5, during adult period. Examination of the topographical distribution throughout the antrum of these two populations shows that, strip per strip, their numerical ratio varies. Homogeneous values for the latter occur in the middle part of antrum and, as rule, in each group they reflect the mean value calculated for the whole of the antrum.


A comparison of receptive and non-receptive plasma membrane areas of photoreceptor cells in the leech, Hirudo medicinalis. Microvillar (receptive) and external (non-receptive) portions of the plasmalemma of photoreceptor cells of Hirudo were compared electron microscopically in thin sections and freeze-fracture replicas. A morphometric approximation showed that the surface area of the microvillar membrane is about 19 times larger than that of the external membrane. The microvillar membrane most probably undergoes extensive membrane turnover. In both segments of the membrane the particles associated with the P- and the E-fracture faces are randomly distributed except at some specific sites. The particles adhere predominantly to the P-faces. The particle densities on the fracture faces of the microvillar membrane differ from those of the external membrane. The P-face particles of the external membrane appear to be larger than those of the microvillar membrane. It is suggested that the P-face particles of the microvillar membrane represent sites where the photopigment is incorporated into the membrane. The distinguishing structural features correspond to the functional differences postulated for both portions of the plasma membrane.


A new type of synapse in the ventral cochlear nucleus. In this paper we report the appearance of flat vesicle-containing endings in aldehyde-fixed ventral cochlear nucleus of rats with qualitative and quantitative properties suggesting they should be identified as calyceal processes. Their synaptic vesicles are elongate and significantly smaller than the vesicles in the calyces of Lenn and Reese (1966). Therefore these endings are flat vesicular calyceal processes, possibly of inhibitory function.


Control of the pars intermedia of the lizard, Anolis carolinensis. II. Ultrastructure of the intact intermediate lobe. The ultrastructure of the intermediate lobe of the hypophysis was studied in Anolis carolinensis with the use of a threefold aldehyde fixative. Lizards with a brown skin were selected. The possibility of two types of secretory cells is discussed; neither cell type is innervated. Type I cells are rarely found and contain dense granules approximately 0.3 micron in diameter; Type II cells vary widely in secretory activity. Most of the Type II cells contain a large number of dense secretory granules (up to about 1.3 micron in diameter) almost filling the cytoplasm. Rough endoplasmic reticulum (RER), Golgi apparatus and mitochondria are poorly developed. Only some of these cells show signs suggesting a high secretory activity, namely a well developed RER, Golgi apparatus and numerous mitochondria. In these cells the RER sometimes forms large intracisternal droplets (up to 7 micron in diameter). Two of the animals exhibited a more uniform, high secretory activity. Large (about 2 micron in diameter), pale vacuoles, probably of extracellular character, were found mostly in the vicinity of the perivascular septum. Their role in the release of MSH is discussed. The present data, which are discussed with reference to earlier findings (Forbes, 1972), form the morphological basis for an experimental study on regulation of MSH release (Larsson et al., 1979).


Synaptogenic effect of estrogen on the hypothalamic arcuate nucleus of the adult female rat. In order to examine the effect of estrogen on the synaptic structures in the hypothalamic arcuate nucleus (ARCN), semi-quantitative studies were performed by counting synapses in an 18,000 micron 2 area in the middle part of the ARCN in each brain. In ovariectomized female rats injected with 2 micrograms of estradiol benzoate (EB) for three weeks, the mean numbers of axodendritic and axosomatic synapses were not significantly different from those in the intact and ovariectomized controls. When the medial basal hypothalamus (MBH) including the ARCN was isolated by use of a Halász knife (MBH island), the mean number of axodendritic synapses was decreased to about half of the controls. However, EB treatment for three weeks from the day of surgery effectively restored the axodendritic synaptic population of the deafferented ARCN. This may suggest that estrogen has a facilitatory effect on axodendritic synapse formation in the deafferented ARCN, presumably by stimulating axonal sprouting and synaptic regeneration of intact axons in the MBH island.


Estrogen target cells in the pituitary of platyfish, Xiphophorus maculatus. The distribution of estradiol-concentrating cells in the pituitary of the platyfish, Xiphophorus maculatus, is studied after the injection of 3H estradiol-17 beta by thaw-mount autoradiography. Autoradiograms prepared 2-8 h after the injection show nuclear concentration of radioactivity in certain cells of the rostral pars distalis, pars intermedia and pars nervosa. Radioactively labeled cells are identified as gonadotropes by a combined technique of thaw-mount autoradiography and immunoperoxidase staining with antiserum to ovine LH. Approximately 80% of the immunoreactive LH cells show a concentration of radioactivity in their nuclei. These observations indicate that in teleosts, as in mammals, estradiol has a direct effect on pituitary gonadotropes.


Fine structure of the transitional zone of the rat seminiferous tubule. An electron microscopic study was made on the structure of the testicular transitional zone (TZ) in the adult rat. The TZ proper consists of modified Sertoli cellss, with only a few spermatogonia and macrophages, surrounding distally a very narrow lumen. The TZ Sertoli cells have nuclei with a somewhat coarser matrix and more peripheral heterochromatin than Sertoli cell nuclei of the nearby seminiferous tubules, and the electron density of the cytoplasm varies from cell to cell. Smooth endoplasmic reticulum is abundant, but usually there are also scattered ribosomal rosettes and an occasional profile of rough endoplasmic reticulum. Microtubules are very numerous in the columnar portion of the cell, and laminar structures seemingly joining the cell surfaces are sometimes seen. Lipid droplets and lysosmal structures are frequent cellular components in proximal TZ Sertoli cells. Empty intracellular vacuoles are abundant, sometimes arranged around areas of smooth endoplasmic reticulum. Occasionally, membrane-limited fine granules and vacuoles are seen within Sertoli cells and also in the TZ lumen, suggesting a possible secretory activity by these cells. The apical processes of the Sertoli cells form large vacuolar structures, and in the basal parts of the epithelium vacuoles with capillary-like appearance are frequently seen. Phagocytosis of germinal cells by the Sertoli cells occurs in the proximal region of the TZ. Round waste bodies in contact with the Sertoli cell apices protruding into the tubulus rectus, are also common. The tunica propria of the TZ is thickened and somewhat wrinkled, and in the proximal region the myoid cell layer loses its continuity and is replaced by fibroblasts. The epithelium of the tubulus rectus adjacent to the TZ consists of several overlapping epithelial cells. The typical junctional complexes between TZ Sertoli cells appear to be impermeable to the lanthanum tracer.


Embryonic development and secretory differentiation in the pars tuberalis of the mouse hypophysis. The pars tuberalis (PT) of the mouse, like that of other mammals, consists mainly of glandular cells rich in glycogen and peculiar to this lobe. In the mouse, the glandular cells are characterized by large, dense secretory vesicles (up to 300 nm in diameter), the abundance of which indicates a marked secretory activity. The PT develops from a distinct antero-ventral area of Rathke's pouch. The border between the anlagen of the PT and the pars distalis is formed by Atwell's recessus which represents the access for the vessels afferent to the pars distalis. The pedicle of Rathke's pouch is incorporated into the PT anlage, thus contributing to its formation. The entire PT anlage is characterized by glycogen accumulation from the commencement of its formation and persisting in the adult tuberal lobe. Secretory differentiation of the glandular cells of the PT occurs at day 12 of gestation, preceding that of all other adenohypophysial cell types. The secretory features of these cells (development of ergastoplasm and Golgi apparatus, abundance of dense secretory vesicles) appear at an early stage of the enbryonic life (14 days) comparable to those of mature cells. These results confirm earlier observations in the foetal rat where hypophysial secretion also begins in the PT. The existence of peculiar glandular cells speaks in favour of a specific but still unknown function of the PT during foetal and adult life.


Formation of sarcomeres in the embryonic heart of the lobster. Cardiomyoblasts in the myocardium of embryonic lobsters at 3-4 weeks and 6 months of development were examined with the transmission electron microscope in order to describe the events in the formation of sarcomeres in a neurogenic cardiac system. Thick and thin myofilaments appear first in the cell periphery near the sarcolemma. They align in parallel in a sequential fashion to form consecutive sarcomeric units. Well-defined A and I bands appear before any semblance of a Z line is present. The initial sarcomere is anchored to the sarcolemma by the insertion of thin myofilaments into a region of electron dense material associated intimately with the sarcolemma. Myofibrils grow outward in several planes away from the electron-dense regions of membrane that serve as focal points for myofibril formation.


Vascular organization of the catfish gill filament. Light and scanning electron microscopic observations were made on methyl-methacrylate corrosion casts of the blood vessels in the gills of channel catfish (Ictalurus punctatus). The vasculature of the gill filament can be divided into three distinct pathways: 1. the well-known respiratory circulation which includes the afferent filamental artery (AF), afferent lamellar arteriole (AL), lamella (L), efferent lamellar arteriole (EL) and efferent filamental artery (EF), 2. a nutritive pathway from the EF through small nutritive capillaries (NC) and into one of several filamental veins (FV), and 3. an interlamellar circulation in which small prelamellar arterio-venous anastomoses (PAVA) connect the AL into a series of organized vascular spaces (interlamellar vessels, ILV's) that underlie the interlamellar filamental epithelium. Several sinus like spaces associated with AF, EF and the filamental cartilagenous support were also observed. The physiological significance of these vascular pathways is discussed.


Membrane specializations in the peripheral retina of the housefly Musca domestica L. Membrane specializations of the peripheral retina of the housefly (Musca domestica) are revealed in thin sections and freeze fracture/etch replicas. Septate junctions are abundant in corner areas of the pseudocone enclosure bonding: between homologous corneal pigment cells (CPC); between homologous large pigment cells (LPC); between CPC-LPC; between Semper cells (SC); between SC-CPC. Spot desmosomes are present between Semper cells. It is likely that septate junctions function as strengthening adhesions in this area. A new membrane specialization similar to a continuous junction was observed between retinular cells of the same or adjacent ommatidium. This junction has indistinct septa in the 115 A intermembrane cleft and is intermittent in character. When this junction is absent, the apposed cells gape apart. In freeze fracture studies, this junction is characterized by bridges composed of fused membrane particles and randomly arranged particles on the P face, and noncorresponding grooves on the E face. The ridges are elongate and roughly parallel and sometimes they form enclosures. Mitochondria line up along these junctions, often within 90 A of the unit membrane. This membrane specialization has characteristics of tight and continuous junctions. In line with previous findings, we suggest that this junction assists in retinular cell orientation, possibly in enforcing the ommatidial twist and in maintaining localized ionic concentration gradients between retinular cells.


The effect of L-DOPA on the ultrastructure of the adenohypophysis of the Chinese quail, Excalfactoria chinensis. Intraperitoneally injected L-DOPA was found to stimulate strongly the lysosomal system of all of the cell types of the adenohypophysis. We suggest that this amine may play a role in the regulation of the secretory activity of the gland by mediating the lysosomal activity, this role being of variable importance according to the fluctuations in hormonal activity. Utrastructural changes occur in some cell types following injection of L-DOPA. The question as to whether these changes are induced directly by L-DOPA or via the monoaminergic system of the hypothalamus and its hypophysiotropic hormones is discussed.


Development of the basal lamina and extracellular materials in the early chick embryo. Chick embryos at developmental stages up to primitive streak formation were fixed in a mixture of tannic acid and glutaraldehyde. A basal lamina was present in the unincubated embryo and consisted of a lucent lamina interna and a lamina densa. At the primitive streak stage the lamina densa showed a periodicity of stained elements. Densely stained materials were present on the cell surfaces lining the cavity between the epiblast and endoblast, and on the mesoderm cells within this cavity. Considerable amounts of extracellular material were observed in the cavity. Hyaluronidase treatment removed the cell surface and extracellular material, indicating that hyaluronic acid is a major component. This enzyme disrupted the basal lamina, leaving a fibrillar remnant with  no periodic structure. It is therefore suggested that the dense periodicities consist of glycosaminoglycan built on an enzyme-resistant framework which is probably collagen. Enzyme-resistant fibrils, presumably collagen precursors, are present elsewhere within the tissue spaces.


The hypothalamo-hypophysial system in the ground squirrel, Citellus erythrogenys Brandt. II. Seasonal changes in the classical neurosecretory system of a hibernator. Monthly observations of the "Gomori-positive" hypothalamo-hypophysial neurosecretory system (HHNS) of the ground squirrel, Citellus erythrogenys Brandt, were carried out light microscopically using several quantitative methods. From the beginning of hibernation, formation of neurosecretory material (NSM) in the neurosecretory cells (NSC) progressively decreases and release of neurohormones from the HHNS is almost fully inhibited. A maximal accumulation of NSM in the perikarya of the NSC and in the posterior pituitary (PP) is found in December. By this time the volume of the cell nuclei and nucleoli is at a minimum. Signs of activation of the HHNS appear and become more conspicuous as the time of arousal from torpor approaches. The amount of NSM in the NSC and the PP decreases simultaneously with the increase in volume of the NSC. Hyperemia and activation of glial elements is visible throughout the HHNS. The morphological signs of activation reach their peak in March. After reproduction is completed (April to beginning of May), the NSC and the PP are almost devoid of NSM. Beginning with June and during the summer and autumn months a progressive accumulation of NSM in the NSC and the PP parallels gradual diminution in the volume of the NSC structures and the glial cells. Mechanisms and effector pathways by which the HHNS influences seasonal adaptation of the organism and reproduction are discussed.


Control of the pars intermedia of the lizard, Anolis carolinensis. III. Changes in the ultrastructure of the disconnected neuro-intermediate lobe. Morphological changes in the disconnected neuro-intermediate lobe were studied in the lizard, Anolis carolinensis from the 2nd to the 14th post-operative day using a threefold aldehyde fixative (Rodríguez, 1969). Two phases of colour change capacity were exhibited: Phase I started immediately after the transection, lasted for 6 days (mean) and was characterised by an excessive MSH release (brown skin). This phase proceeded gradually into Phase II, designated by an interruption by the MSH release (green skin). The degenerative processes and final elimination of neurons in the disconnected neural lobe propagate in a rostro-caudal direction from the transected area. The aminergic fibres (Type II) disappear within 2 days postoperatively, whereas the degeneration continues for more than 10 days in the peptidergic fibres (Type III, IV and V). The glia cells (ependyma and pituicytes) serve as very active macrophages, engulfing fragments of axons already affected by autolysis and transferring them into glial lysosomes. No apparent morphological changes occur in the shift from Phase I to II. The great majority of the secretory cells of the intermediate lobe are not affected by degenerative processes and appear to be markedly activated by the stalk transection. They exhibit numerous mitochondria, well-developed Golgi complexes forming numerous Golgi vesicles and extensive parallel cisternae of the rough endoplasmic reticulum, sometimes forming large intracisternal droplets (7 micron in diameter). Numerous pale vacuoles are seen, especially toward the intact capillaries, suggesting their coupling to the MSH release by extension of the active membrane area toward the perivascular septum. The number of these vacuoles is very markedly reduced in Phase II (no release), whereas the formation of new granules seems to proceed in early stages. The interruption of the MSH release implies a successive refilling of gradually growing secretory granules and a concomitant reduction in the development of the synthetic apparatus. Mechanisms probably involved in the control of the synthesis and release of MSH are discussed.


An ultrastructural and cytochemical study of the interaction between latex particles and the haemocytes of the wax moth Galleria mellonella in vitro. The plasmatocytes are the major phagocytic blood-cell type in the haemolymph of the wax-moth, Galleria mellonella. In the present study, these cells were allowed to attach to tissue culture dishes for 1 h, rinsed and then incubated with latex beads for up to 72 h. These cells were then fixed for routine transmission electron microscopy and acid phosphatase cytochemistry. Intracellular latex particles were found in tight, ill-defined phagosomes, which were often clearly associated with the Golgi complexes of the plasmatocytes. Fusion of both primary lysosomes and multivesicular bodies with the phagosomes occasionally occurred and this resulted in the accumulation of an acid phosphatase positive reaction product around the test particles. Subsequent experiments showed that this acid phosphatase activity was mainly associated with the primary lysosomes. The results of the lysosome/latex interactions are compared with those obtained from similar studies on the digestive mechanisms in other phagocytes.


A cytochemical and immunofluorescence study of endocrine cells in the gut of the ascidian Styela clava. Strong secretin-like immunofluorescence has been demonstrated in endocrine-like cells from the gastric epithelium of Styela. These cells also stain with lead haematoxylin and exhibit a brilliant formaldehyde-induced fluorescence, but do not show any other cytochemical features characteristics of the mammalian APUD series. Tests with antisera to glucagon, gastrin and somatostatin all proved negative. In the oesophagus tests with all four antisera proved negative. The significance of these results is discussed in relation to the phylogeny of vertebrate gastro-intestinal hormones.


Calcium binding sites in the vesicles of the carotid and aortic body chief cells. Chief cells of the carotid and aortic body chemoreceptors possess numerous cytoplasmic dense-core vesicles which are known to contain primarily dopamine. Following fixation in solutions containing 50 mM CaCl2, a 20--30 nm electron-dense particle (EDP) is often observed eccentrically located in many of the vesicles. Approximately 44% of the carotid body and 16% of the aortic body vesicles contain an EDP. The EDP probably represents the Ca++ binding site critical to the stimulus-secretion coupling events culminating in exocytosis of these vesicles. The presence of Ca++ in the cytoplasmic vesicles was verified by electron probe X-ray microanalysis.


Cell-to-cell contacts in primary cultures of dissociated chicken embryonic brain. The fine structure of intercellular contacts was studied in primary cultures prepared from chicken embryonic brain. Desmosomes were frequently seen between the glial cells. Synaptic contacts were observed among neuronal cell bodies and neural processes after 8 days in vitro. Gap junctions were revealed between glial elements suggesting a functional role in direct cell communication and providing a morphological basis for previous observations on potassium transport in cultures of dissociated brain cells.


Neuro-epithelial bodies in organ cultures of fetal rabbit lungs. Ultrastructural characteristics and effects of drugs. Lung explants from fetal rabbit at the late glandular stage of development (20 days' gestation) and near term (31 days' gestation) were maintained in organ culture for up to 22 days. They were studied by light and electron microscopy to determine whether neuro-epithelial bodies (NEB) of the lung retain structural integrity in vitro. Cultured NEB retained argyrophilia and specific amine fluorescence after formaldehyde condensation. Their ultrastructural morphology showed some differences from that of uncultured NEB: the terminal axons had degenerated and the secretory granules (dense-core vesicles, DCV) were slightly larger, more pleomorphic, more electron-dense, and redistributed throughout the cytoplasm rather than being confined chiefly to the basal regions. These changes, together with hypertrophy of Golgi zones, suggest increased synthesis and storage of secretory products in the DCV during culture. In NEB from near-term explants cultured for 7 days and incubated with reserpine, the core of DCV decreased in size and electron-density and became finely granular, a sign of amine release. Ca++ ionophore No. A-23187, also, induced changes in the ultrastructure of DCV, suggesting that the secretory process in lung neuro-endocrine cells, as in other secretory cells, is Ca++-dependent.


Spatial distribution of excitatory innervation on a muscle fiber of the lobster (Homarus americanus). The distribution of nerve terminals from a single excitatory motor axon has been followed along a fiber from the proximal accessory flexor muscle of the lobster by thin serial sectioning at periodic intervals. The excitatory motor axon provides small axonal branches (diameter 5--12 micron) of varying lengths that travel along the surface of the muscle fiber. Each of these branches gives rise to discrete synaptic terminals which are not uniformly distributed. Individual terminals vary in length from 10--60 micron and the majority possess neuromuscular synapses. The greatest distance between synapses is slightly over 1 mm which is well within the length constant (2.6 mm) of this muscle fiber. Thus the spatail distribution of synapses is such as to ensure adequate depolarization along the entire length of the muscle fiber.


Ultrastructural studies on erythropoiesis in the avian thymus. II. A stereological analysis of the lymphoid and erythroid cells. The cortex of enlarging thymic lobes from adult haemorrhaged Quelea quelea were found to be similar to those of wild birds where the thymic enlargement was occurring naturally. A detailed stereological analysis of cells broadly designated as lymphoid, and the construction of models to account for the results, indicates that the enlarging thymic lobe contains both large and small blast cells, a heterogenous group of medium lymphocytes, erythroid cells, and two types of very small lymphocytes. The distinction between early erythroid cells and some lymphocytes, despite this detailed analysis is very difficult, but it is possible in enlarging thymic lobes that up to 42% of the lymphoid cells may have erythroid characteristics.


Ultrastructural studies on erythropoiesis in the avian thymus. I. Description of cell types. Thymus lobes from three species of birds, Quelea quelea, Passer domesticus and Sturnus vulgaris, have been examined ultrastructurally. The component cell types are compared with their counterparts in mammalian thymus glands, and found to be similar. Greater differences between small, intermediate and enlarged lobes of one species than exist between species. Developing erythroid cells are present in most enlarging and some enlarged glands. They appear to be developing at the expense of lymphoid cells in some birds. The origin of these cells is discussed. Cells that are possible candidates for the production of some thymic hormones are also described.


Ionic gradients within mantle-collar epithelial cells of the land snail Otala lactea. Aestivating Otala lactea have been shown to reduce the rate of evaporative water loss from the cells of the mantle-collar epithelium to a level comparable to that of an insect. X-ray microanalyses of ultrathin frozen sections from aestivating and non-aestivating snails have shown gradients of chloride and potassium ions in the apical microvillus region of the regulating mantle collar epithelium. The greatest difference in osmotic concentration occurs in the apical 2 micron of the cell. There appears to be a barrier at that level that prevents water being mobilised from the underlying tissues. Methods of presenting data generated by X-ray microanalysis are also discussed.


Organization and development of the perivascular space system in the neurohypophysis of the laboratory mouse. The organization of the system of perivascular space around the capillaries in the neurohypophysis was studied in the adult and developing laboratory mouse by the use of histological silver impregnation and electron microscopical techniques. In the median eminence short and long extensions, arising mainly from the shallow space around capillary loops of the primary plexus of the portal system, formed radiations into the adjacent neural tissue of the external zone. The tissue of the neural lobe was separable into non-vascular regions dominated by undilated portions of neurosecretory nerve fibres and pituicytes, and neurovascular regions with perivascular space extensions forming an extensive system of connections between neighbouring capillaries. In the median eminence, the system of extensions of the perivascular space was estimated to increase the neurovascular contact surface area by at least 50%, implying an increased efficiency of the organ without a notable increase of its volume. The possibility that the ramifications of the perivascular space imply an enhanced uptake rate into the bloodstream and a subsequent increased concentration of the neurohormones in the portal blood, was discussed. During development of the median eminence, differentiation of perivascular space extensions of the adult type started in the juvenile of about three weeks of age, when shallow capillary loops had been formed. In the neural lobe, perivascular space ramifications were already present when the internal capillaries were formed and were fairly frequent in ten-day young. At the age of three to four weeks the organization of the system was similar to that of the adult animal.


The sea urchin (Sphaerechinus granularis) codes different H2B histones to assemble sperm and embryo chromatin. Histones from sperm and gastrula nuclei of the sea urchin Sphaerechinus granularis were analyzed by three different electrophoretic methods, stained with specific color reactions and compared with one another. The histones of the two cell types showed identical properties in none of the three analytical methods employed but the greatest differences involved the H2B type of histones. H2B from embryo chromatin had properties similar to those of the corresponding molecule from calf thymus. In the sperm chromatin no such molecule was observed but two additional ones were found that differed from embryo H2B in size, charge and specific colour reactions. The differences were such that different genes may be postulated for the synthesis of the H2B histones found in sperm and in embryo.


Metabolic stability of nonhistone chromosomal proteins in two different classes of developing rat brain nuclei. The metabolic behaviour of NHCP was studied in rat brain nuclei from fully differetiated cells and in nuclei from still differentiating cells. Eight-day-old rats were injected intracisternally with [14C]thymidine and [3H]tryptophan and the 3H/14C ratio of the chromatin was followed for a period of 33 days. It was found that in the fraction of differentiated cells this ratio remained unchanged, showing the presence of metabolically stable NHCP. At the same time in the fraction containing nondifferentiated cells a substantial part of the NHCP was turned over, which was indicated by a sharp decrease in their 3H/14C ratio with time.


DNAase hydrolysis of chromatin DNA in intact sea urchin sperm cells. Effect of the ionic strength on the digestion parameters. Chromatin DNA in intact functional Sphaerechinus granularis sperm cells has been digested with micrococcal nuclease at three different ionic strength conditions. The results show a highly-flexible chromatin organization similar to that found in sperm heads. The rate of digestion, the limit value of acid-soluble material and the fragmentation pattern show that the sensitivity of nucleosome and internucleosome DNA regions to nuclease hydrolysis depends on a delicate balance of polar and non polar interactions. At low ionic strength, both nucleosome and internucleosome regions are rapidly and completely hydrolysed at the same time and a transient subunit fragment of 120 b.p. average length is formed. At high ionic strength, internucleosome regions are preferentially hydrolysed; there is a limit digest value and a stable subunit fragment of 140 b.p. average length is formed. A supernucleosome organization in the high ionic strength environment of the sperm cells is suggested by the transient preferential formation of heptamers of nucleosome DNA fragments.


Characterization of presumptive histone messenger RNA from a cell line of Aedes aegypti. Four presumptive histone messenger RNAs were characterized from a cell line of Aedes aegypti, and their molecular weights were determined by electrophoresis. They were shown to be associated with polysomes during the peak of DNA synthesis, but not when DNA synthesis was inhibited by cytosine arabinoside or when DNA was not being synthesized. These mRNAs are associated with polysomes containing less than 8 ribosomes and having a high ratio of incorporation of lysine to tryptophan into their nascent peptides. The mRNAs released from these polysomes were translated in vitro and histone products were synthesized. Histones were not synthesized when the mRNAs were obtained from large polysomes or from small polysomes during the non-DNA synthetic period.


Differentiation without mitosis in Dictyostelium discoideum. Dictyoselium discoideum Ax-2 amoebae incubated in the presence of the microtubule inhibitor nocodazole, irreversibly lost their ability to multiply. Nocodazole-treated cells remained viable and RNA and protein synthesis continued for at least 48 h. When nocodazole-treated amoebae were allowed to develop on Millipore filters or on agar slides they differentiated with some delay when compared with controls. These results show that mitosis, naturally present during the developmental cycle of Dictyostelium discoideum Ax-2, is not indispensible for differentiation.


Ethanol inhibition of vinyl chloride metabolism in isolated rat hepatocytes. Isolated rat liver cells convert [14C]vinyl chloride into non-volatile metabolites. The metabolism is not increased by in vivo pretreatment with phenobarbital. It is sensitive to inhibition by ethanol, which at a concentration of 4 mM inhibits vinyl chloride metabolism to 50% in hepatocyte suspensions. The metabolic activity is NADPH-dependent and is localized in the microsomal fraction of the liver. The enzyme is also strongly inhibited by tetrahydrofuran, indicating that it could be identical to an ethanol-inducible cytochrome P-450 described in the literature [1].


The interaction of cephaloridine with model membrane systems and rat kidney lysosomes. The antibiotic cephaloridine has been shown to interact with phospholipid structures, using the techniques of ultraviolet difference spectroscopy, surface pressure measurements and liposome models. The results indicate that this interaction is at least partly hydrophobic in nature and help explain the disruptive effects of high concentrations of cephaloridine on both artificial and natural phospholipid structures (lysosomes). Low concentrations of cephaloridine were shown to inhibit a lysosomal membrane-bound phospholipase 2 and it is suggested that such an inhibition may explain the cephaloridine-induced stabilization of rat-kidney lysosomes.


Mutagenicity and irreversible binding of the hepatocarcinogen, 2,4-diaminotoluene. Mutagenicity of 2,4-diaminotoluene (DAT) in the Salmonella mutagenicity assay was increased with liver fractions from phenobarbital (PB) or beta-naphthoflavone (BNF) treated rats. Substitutions of the hydrogens in the methyl group of 2,4-DAT with deuterium resulted in a decrease in mutagenicity. Incubation of rat liver microsomes with tritiated 2,4-DAT in the presence of NADPH led to the formation of irreversibly bound products to microsomal protein. The rates of binding were not increased using microsomes from PB or BNF-treated rats and was not altered by deuterium substitution in the methyl group. Addition of superoxide dismutase, glutathione (GSH) or rat liver supernatant reduced 2,4-DAT irreversible binding, whereas 2,4-DAT mutagenicity was unaffected by superoxide dismutase addition. Injection of tritiated 2,4-DAT 100 mg/kg to rats lead to its irreversible binding to liver protein and ribosomal RNA and to kidney protein in vivo, again protein binding was not increased after prior treatment with PB or BNF. No irreversible interaction of tritiated 2,4-DAT with DNA either in vitro or in vivo could be demonstrated.


Binding of 6-hydroxymethylbenzo[a]pyrene and 6-acetoxymethylbenzo[a]pyrene to DNA. The carcinogenic hydrocarbons 6-hydroxymethylbenzo[a]pyrene (6-HOCH2-B[a]P) and 6-acetoxymethylbenzo[a]pyrene (6-AcOCH2-B[a]P) were examined for their ability to bind to rat and calf thymus DNA. The data indicate there are no appreciable differences in the amount of binding to the two types of DNA. Non-enzymatic binding of 6-HOCH2-B[a]P was low (5 mumol hydrocarbon/mol DNA P) but 6-AcOCH2-B[a]P was bound to a considerable extent (88.4--97.3 mumol hydrocarbon/mol DNA P). Non-enzymatic binding of 6-HOCH2-B[a]P was greatly increased in the presence of ATP. Binding of 6-HOCH2-B[a]P in the presence of liver microsomes from untreated rats or from rats pretreated with 3-methylcholanthrene (3-MC) never exceeded 5 mumol hydrocarbon/mol DNA P. Binding of 6-HOCH2-B[a]P in the presence of a PAPS generating system was less than non-enzymatic binding mediated by ATP and was dependent on the presence of ATP rather than ATP and sulfate. Binding was reduced by 50%  when ADP was employed in the non-enzymatic reaction and was negligible in the presence of AMP or adenosine, indicating that a diphosphate group is necessary. Incubation of 6-HOCH2-B[a]P with DNA in the presence of ATP, CTP, GTP, or UTP showed that ATP was the most effective mediator of the binding reaction. These observations suggest that 6-HOCH2-B[a]P is converted to a phosphate ester which, like 6-AcOCH2-B[a]P, is much more reactive than 6-HOCH2-B[a]P itself.


Comparative cytotoxic and antitumoral effects of ellipticine derivatives on mouse L 1210 leukemia. Twelve derivatives of the antitumoral alkaloid ellipticine (E) and ellipticinium were assayed in vitro on cultured L 1210 cells. These drugs possess varying abilities to decrease the cell growth rate in a 1--1000-fold range. Some of them have a highly cytotoxic effect in the 10(-8)--10(-6) M range. Non-specific intracellular damages are produced: multilobation of nuclei, occurrence of numerous lipid granules, diminution of the size and increase in the number of mitochondrial profiles and several modifications of the internal architecture of mitochondria. 2-Methyl-9-hydroxyellipticinium (2-CH3-9-OHE) was submitted to a bioassay; it inactivates the tumorigenic potency of the cells exposed to it, when they are grafted back into mice in the same dose range which reduces in vitro the growth rate of the cells. A fairly good correlation holds between the in vitro and in vivo (antitumor effect) assays, offering a possible prescreening test for a cheaper and rapid evaluation of chemotherapeutic activity of these compounds. The results stress again the importance of the 9-hydroxy substitution in these series for improving the anticancer efficiency. The nature of the biochemical target of E and derivatives is discussed according to our data.


Studies on the interaction between steffimycins and DNA. The complexes formed between steffimycins and DNA were studied using various physicochemical techniques. The binding process has been followed spectrophotometrically or fluorimetrically. The binding parameters n and K, evaluated according to McGhee and Von Hippel, show a good affinity of these antibiotics for the macromolecule. Flow dichroism measurements showed that in the complex with DNA, the antracycline moiety of the steffimycins is intercalated between two base pairs of the macromolecule. The binding experiments with various polydeoxyribonucleotides and with various DNA samples, having different base pair compositions, suggest that an alternate sequence of A-T, such as that of poly[d(A-T)] . poly[d(A-T)], represents a good receptor site for the binding of steffimycins to DNA. The lack of in vivo activity of these antibiotics is discussed.


Interaction of DNA and purine nucleosides with cis-dichlorodiammineplatinum(II) and antimitotic activity of the complexes on meristematic root cells. The appropriate experimental conditions for the preparation of complexes of cis-dichlorodiammineplatinum(II) with DNA and with purine nucleosides have been determined which leave negligible amounts of free drug in the solution. Important conformational changes of DNA upon binding to cis-Pt(NH3)2-Cl2 have been evidenced through viscosity, electric birefringence and thermal denaturation experiments. The antimitotic and antitumor activity of the drug was found to be totally inhibited by its binding to DNA and to the purine nucleosides. Enzymic degradation observations on the DNA-cis-Pt(NH3)2Cl2 complexes indicated an important inhibition of the degradation and the absence of release of free drug. The implications of the results in relation with the mode of binding of this compound to DNA, with the choice of carriers for drugs and the mechanism of action of lysosomotropic agents are discussed.


Isolation and partial characterization of a cadmium-binding protein from the American oyster (Crassostrea virginica). American oysters (Crassostrea virginica) were exposed to 0.1 ppm cadmium for 0--15 days in a flowing seawater system and then placed into clean flowing seawater for 24 h prior to sacrifice. Whole oysters were homogenized and a cadmium-binding protein isolated and purified by a process of centrifugation, heat-treatment, Sephadex G-75 chromatography, DEAE cellulose chromatography and disc gel electrophoresis. A highly anionic protein which is not present in control oysters was found to be present in cadmium-exposed animals after 3 days of treatment and to increase in concentration at succeeding time points. The protein does not extensively bind zinc or copper. Amino acid analysis of the purified protein disclosed an amino acid composition characterized by a high percentage of dicarboxylic amino acids and relatively little cysteine.


Metabolism of benzo[A]pyrene in hamster embryo cells. Effect of the concentration of benzo[A]pyrene on its metabolism. The metabolism of benzo[a]pyrene (BP) by hamster embryo cells was studied. The production of water-soluble metabolites, shown to be conjugates with glucuronic acid, depended on BP concentration. With increased BP concentration the amount of glucuronic acid conjugates increased, but the proportion of conjugates in BP or its metabolites present in the medium decreased. The metabolites extracted with ethylacetate were trans-7,8-dihydrodiol-BP (7,8-dihydrodiol) and trans-9,10-dihydrodiol-BP (9,10-dihydrodiol), but large peaks of phenolic metabolites were found by high pressure liquid chromatography (HPLC) after digesting the medium with beta-glucuronidase. Therefore, BP is metabolized to oxygenated forms, and of these, most of the phenolic metabolites and parts of the dihydrodiols are conjugated with glucuronic acid. The proportions of dihydrodiols to phenols, estimated by HPLC after beta-glucuronidase digestion, decreased when the BP concentration was decreased. The results suggest that dihydrodiols are less readily glucuronidated than phenols and so may be metabolized further to metabolites other than glucuronic acid conjugates.


Binding of products originating from the peroxidation of liver microsomal lipids to the non-lipid constituents of the microsomal membrane. The binding of products derived from the peroxidation of liver microsomal lipids to the non-lipid constituents of the microsomes was studied. To this end arachidonic acid labelled with tritium at the positions of the double bonds was given to rats and allowed to incorporate into the membrane lipids of the liver cell. When liver microsomes containing labelled arachidonic acid were incubated aerobically in the NADPH-dependent system, a marked production of malonic dialdehyde (MDA) occurred and, concomitantly, there was a consistent release of radioactivity from the microsomes into the incubation medium. The addition of EDTA to the incubation medium prevented, to a large extent, both the MDA formation and the release of radioactivity. Chromatographic studies showed that the bulk of the radioactivity released from the incubated microsomes is not MDA. In the incubated microsomes, the radioactivity decreased in total lipids, while it increased by about 15 times in the non-lipoidal residue. A similar increase in radioactivity was seen in microsomal protein, while no increase was observed in microsomal RNA (the radioactivity was negligible in both the incubated and the non-incubated samples). It seems therefore that products originating from lipoperoxidation of arachidonic acid covalently bind to the microsomal protein. In order to investigate whether alterations similar to those observed in the in vitro peroxidation of liver microsomes could be detected in the in vivo intoxication with carbon tetrachloride, rats given labelled arachidonic acid as above, were poisoned with CCl4. Sixty minutes after poisoning, the radioactivity present in the microsomal lipids was generally lower in the intoxicated rats than in the controls, while the labelling of the non-lipoidal residue and of the protein was higher in the CCl4-poisoned rats.


The effects of 1,1-di(p-chlorophenyl)-2-chloroethylene on plasma enzymes and blood constituents in the Japanese quail. Glutamate oxaloacetate transminase (GOT), glutamate dehydrogenase (GDH), sorbitol dehydrogenase (SDH), pseudo-cholinesterase (ChE) and various blood constituents were measured in the plasma of Japanese quail fed 1,1-di(p-chlorophenyl)-2-chloroethylene (DDMU) at low levels for periods ranging from 2 to 32 days. Previous work has shown that DDMU is a potent inducer of hepatic microsomal enzymes causing marked structural changes in the liver. A rapid increase in plasma GOT was observed within 4 days accompanied by an increase in relative liver weight. Plasma GDH and SDH increased to a maximum between 16 and 24 dyas which seems to be associated with hepatic cell proliferation. Plasma ChE showed a steady increase over the time course of DDMU administration. The level of plasma lipid was reduced after 4 days whereas the hepatic lipid content was substantially increased suggesting that the fatty liver condition may be caused by decreased release of triglyceride from the liver. Plasma glucose was reduced at 8 days but there was no evidence of a hyperglycaemic state. The changes noted after 2 days of DDMU diet were confirmed by measurements on birds 18 h after oral dosing the DDMU. The study demonstrates the value of plasma enzyme measurements for the early detection of toxic effects and indicates that DDMU administration leads to extrahepatic effects in addition to those previously described in the liver.


Persistence and accumulation of (potential) single strand breaks in liver DNA of rats treated with diethylnitrosamine or dimethylnitrosamine: correlation with hepatocarcinogenicity. Effects of diethylnitrosamine (DEN) and dimethylnitrosamine (DMN) on the sedimentation pattern of [3H]thymidine-labelled Sprague-Dawley female rat liver DNA in alkaline sucrose gradients were studied with regard to time and dose dependency. In experiments at 1--56 days after a single injection it was observed that (potential) single strand breaks induced by DEN were repaired at a low rate. At 56 days the sedimentation pattern was still grossly abnormal. Half-life values of 27 and 46 days were observed after 134 mg/kg DEN (approx. 45% of the LD50) and 13.4 mg/kg DEN, respectively. Identical experiments after DMN (10 mg/kg, corresponding to about 35% of the LD50) showed return to (almost) completely control sedimentation patterns within 56 days after injection (t 1/2 = 8 days). Experiments at 6 or 56 days after the last of a series of 5 or 10 weekly injections of DEN (13.4 mg/kg) showed that a major part of DEN-induced damage (measured as single strand breaks) is of a persistent and accumulating character. No accumulation of DMN-induced rat liver lesions was observed. It is concluded that DNA fragmentation and lack of DNA repair is not a consequence of hepatotoxicity. Since at equimolar doses DEN gives appreciably less DNA alkylation (including O6-alkylguanine) but is much more effective both as an inducer of preneoplastic liver lesions and as a hepatocarcinogen when compared with DMN, we believe that the formation of persistent (and accumulating) DNA damage after DEN administration might be relevant in the process of liver tumour formation.


Studies on the distribution and metabolism of N-[14C]nitrosopyrrolidine in mice. Pretreatments with pyrazole, ethanol, nialamide or diethyldithiocarbamate were found to strongly depress the exhalation of 14CO2 and the incorporation of radioactivity in the acid-insoluble fraction of the liver in mice injected with N-[14C]nitrosopyrrolidine. Whole-body autoradiography performed with hemisections of mice at -80 degrees C (to prevent evaporation of the volatile N-nitrosopyrrolidine) and with dry tape-sections (to localize the non-volatile metabolites), using pretreated and non-pretreated mice, indicated a uniform distribution of the non-metabolized N-nitrosopyrrolidine in the tissues. At the shortest survival intervals (1 and 5 min), a high level of metabolites were found in the liver, the tracheo-bronchial and nasal mucosa and Harder's gland, indicating a local formation of metabolits in these tissues. At later survival intervals (0.5--24 h) metabolites were in addition found in tissues with a rapid cell turnover and a high rate of protein synthesis and in brown fat, which probably reflects incorporation of metabolites via normal biosynthetic pathways. Autoradiography of N-[14C]nitrosopyrrolidine in mice given the substance orally resulted in distribution pictures similar to those obtained after i.v. injections.


Preferential S-sulfonate formation in lung and aorta. S-sulfonate (S-SO-3) compounds have previously been identified as metabolites of sulfite in the plasma of several species of mammals [6--8]. We now report the formation of non-diffusible and relatively stable S-sulfonates in the aorta and lung lobes of rabbits exposed intravenously to constant arterial sulfite concentrations of approx. 550 microM. Under these conditions the kinetics of S-SO-3 formation were first order with coefficients in the range of 0.3--0.4 h-1 and asymptotic concentrations of approx. 900 and 9000 nmol S-SO-3/g dry wt. of lung and aorta respectively. The kinetics of this reaction in aorta tissue were closely approximated in vitro. Clearance of S-SO-3 from both lungs and aorta appeared to be first order with a half-life of 2--3 days.


Epoxide hydrase and glutathione S-transferase activities with selected alkene and adrene oxides in several marine species. Epoxide hydrase and glutathione (GSH) S-transferase activities were measured in subcellular fractions prepared from liver or hepatopancreas and some extrahepatic organs of a number of marine species common to Maine or Florida. These activities were easily detected in the species studied. In fish, hepatic GSH S-transferase activities were normally higher than hepatic epoxide hydrase activities for the alkene oxide (styrene oxide and octene oxide) and arene oxide (benzo[a]pyrene 4,5-oxide) substrates studied, whereas in crustacea, hepatopancreas epoxide hydrase activities were higher than hepatopancreas GSH S-transferase activities with the same substrates. Extrahepatic organs from fish and crustacea usually had higher GSH S-transferase activities than epoxide hydrase activities with the alkene and arene oxide substrates. GSH S-transferase activity was also found in liver or hepatopancreas of every aquatic species studied and in a number of extrahepatic organs, when 1,2-dichloro-4-nitrobenzene or 1-chloro-2,4-dinitrobenzene served as substrate.


In vivo alkylation of foetal, maternal and normal rat tissue nucleic acids by 3-methyl-1-phenyltriazene. The carcinogen 3-methyl-1-phenyltriazene (MPT) was administered subcutaneously to normal or pregnant BD VI rats and DNA and RNA were isolated from various tissues after 8 h or 15 h, respectively. Sephadex G-10 chromatography of DNA hydrolysates showed the presence of 7-methylguanine in all tissues examined including that of the brain, one of the target organs for tumour induction. The amounts of the minor product, O6-methylguanine, were characteristic of an SN1 reaction mechanism. Dowex-50 chromatography of RNA hydrolysates showed the presence of 7-methylguanine and of the minor product, 3-methylcytosine. The relative amounts, both of the methylated bases in the individual nucleic acids and of 7-methylguanine in DNA and RNA, were similar to those found previously after administration of 3,3-dimethyl-1-phenyltriazene (DMPT). This suggests the involvment of a common alkylating intermediate. De novo incorporation of radioactivity into purine bases was detected in both DNA and RNA although the levels were not related to the amounts of methylation. The results show that MPT is sufficiently stable to alkylate nucleic acids in vivo and are consistent with the hypothesis that this reaction is a prerequisite for tumour induction. Futhermore, they support the proposal that MPT is the active intermediate in the induction of tumours by DMPT.


Interaction of benzo [A] pyrene and a hyperplastic agent in epidermal nuclear enlargement in the mouse. A dose response study. Experiments were conducted on the effects of various dose levels of benzo [a]pyrene (BP) on nuclear size in mouse interfollicular epidermis over a 3-day period. Topical application of BP was made with or without croton oil (CO) (0.1 or 0.5%) in the vehicles acetone, toluene and methyl ethyl ketone (MEK). Nuclear size was measured on histological sections either manually or by Quantimet Image Analyser. Vehicle controls treated with 0.1 or 0.5% CO in acetone or MEK gave rise to epidermal hyperplasia with some nuclear enlargement and toluene without CO produced a similar response. It was found that when BP was applied in a vehicle capable of inducing hyperplasia, the nuclear enlargement produced was greater than that produced by either the vehicle control or BP in a non-irritant vehicle. The enhancement of response to BP when tested in the presence of a hyperplastic agent resulted in lower concentrations of BP being detectable. As the levels of BP detectable by nuclear enlargement under these conditions compared reasonably well with those detectable in long-term tests, this system might be usable as a basis for a short-term test for carcinogens.


Dose and effect of methyl-2-benzimidazolylcarbamate in the "mammalian spot test", an in vivo method for the detection of genetic alterations in somatic cells of mice. In the spot test, mouse embryos which are heterozygous for four different recessive coat-colour genes are treated in utero by injection of a mutagen into the peritoneal cavity of the mother or by other appropriate routes of administration. If this treatment leads in a pigment precursor cell to an alteration of the wild type allele of one of the genes under study or to its loss, a colour spot in the adult coat may be seen. Peroral application of 100-300 mg methyl-2-benzimidazolylcarbamate (MBC)/kg to the mother during the tenth day postconception led to an increase in the frequency of colour spots in the coats of offspring. The data are consistent with the hypothesis that MBC is a point mutagen.


Reactions of the carcinogen N-acetoxy-4-acetamidostilbene with nucleosides. The carcinogen N-acetoxy-4-acetamidostilbene (N-AcO-AAS) yields multiple products in reactions with guanosine, adenosine or cytidine in aqueous acetone. The major product from the reaction with cytidine is a deamination product, 1-(4-acetamidophenyl)-1-(3-uridyl)-2-hydrosy-2-phenylethane. Three minor products were unstable and were characterized only by their UV spectra and pK values. Adenosine yielded two major products, one of them 1-(4-acetamidophenyl)-1-(N6-adenoxyl)-2-hydroxy-2-phenylethane, and the second 3-(beta-D-ribosyl)-7-phenyl-8-(4-acetamidophenyl)-7,8 dihydroimidazo [2,1-i] purine. The major adduct with guanosine is 1-(4-acetamidophenyl)-1-(1-guanosyl)-2-hydroxy-2-phenylethane. One minor adduct also appears to be a guanosine-N-1 derivative, while two other minor adducts yield 1-(4-acetamidophenyl)-2-phenyl-1, 2-ethanediol on acid hydrolysis, and thus appear to be O6-derivatives. None of the guanine adducts isolated had the properties of N-7, C-8 or N2 adducts. In this respect, N-Aco-AAS appears to behave more like a classical alkylating agent than like previously studied N-acetoxy-N-arylacetamides, although the target organs of 4-acetamidostilbene are the same as those of other N-arylacetamides.


Reactions of the carcinogen N-acetoxy-4-acetamidostilbene with polynucleotides in vitro. N-Acetoxy-4-acetamidostilbene (N-AcO-AAS) has been shown to react with mononucleosides to give numerous alkylation products [1]. In this work, homopolynucleotides, RNA and DNA were treated with N-[beta-14 C]-AcO-AAS, washed, degraded with S1 nuclease and acid phosphatase, and chromatographed on Sephadex LH-20. RNA prepared in vitro with 14C on cytosine, adenine or guanine was treated with non-radioactive N-AcO-AAS, then digested and chromatographed similarly. By this means, many of the adducts rising from nucleoside reactions were shown to result from treatment of nucleic acids with the same carcinogen, as well as a number of products which have not been matched to products of monomer alkylation. Labeled 1-(4-acetamidophenyl)-2-phenyl-1, 2-ethanediol was detected in the digest of RNA treated with radioactive N-AcO-AAS, suggesting that phosphate alkylation had taken place.


Comparison of aflatoxin B1 and aflatoxin G1 binding to cellular macromolecules in vitro, in vivo and after peracid oxidation; characterisation of the major nucleic acid adducts. A comparison between [14C]aflatoxin B1 (AFB1) and [14C]aflatoxin G1 (AFG1) binding to rat liver and kidney cellular macromolecules has shown AFG1-DNA and-ribosomal RNA binding to be lower in both organs. For both mycotoxins more was bound to nucleic acids than to protein. Two hours after intraperitoneal injection (60 microgram/100 g) of [14C] AFB1, 40 ng, 151 ng/mg. Loss of radioactivity bound to liver DNA for both [14C]AFB1 and protein respectively and for [14C]AFG1 the respective figures were 10, 7 and 1 ng/mg. Loss of liver bound radioactivity to DNA for both [14C]AFG1 and [14C]AFG1 appeared to be biphasic indicating that an enzymic DNA repair process may be operating. In vitro binding studies also showed less AFG1 was bound to exogenous DNA after microsomal activation than AFB1. This difference was not a result of differences in the chemical reactivity of the "ultimate" electrophilic species, the respective expoxides, since chemical activation studies using 3-chloroperbenzoic acid showed similar amounts of AFG1 and AFB1 to be converted to the epoxides and to bind to DNA. Studies on the distribution coefficients of the two mycotoxins showed AFB1 to be more lipophilic than AFG1 and this may be an important factor in determining the weaker carcinogenicity of the latter compound. Characterisation of the major AFG1-DNA adduct formed in vitro, in vivo and after peracid oxidation showed it to have the structure trans-9,10-dihydro-9-(7-guanyl)-10-hydroxy-aflatoxin G1. This adduct is similar to that obtained from AFB1 by activation in vivo, in vitro and after peracid oxidation.


Quantum chemical studies of polycyclic aromatic hydrocarbons and their metabolites: correlations to carcinogenicity. In the context of the bay region hypothesis for polycyclic aromatic hydrocarbon (PAH) carcinogenesis, molecular properties were calculated for seventeen polycyclic aromatic hydrocarbons related to (1) intrinsic substrate reactivities towards activating and detoxifying metabolism and (2) the stabilities of the putative carbocation ultimate carcinogens. All-valence electron methods were used, avoiding the inherent difficulties found in the pi-electron methods. The calculated substrate reactivities were found to predict major metabolites successfully, supporting the validity of their use in attempted correlations with observed carcinogenic potencies. Positive correlations were found between observed carcinogenic potencies and (1) the reactivities of the parent polycyclic aromatic hydrocarbons towards the initial distal bay region epoxidation and (2) the stabilities of the diol epoxide carbocations. The reactivities of the distal bay region diol epoxides, were high for both carcinogenic and non-carcinogenic compounds, implying that the second epoxidation does not determine relative carcinogenic activity. Support for a possible alternative hypothesis, that polycyclic aromatic hydrocarbons are activated by one electron oxidation, was also found.


The action of rat cytosol enzymes on some methylated nucleic acid components produced by the carcinogenic N-nitroso compounds. The stabilities of several alkylated nucleic acid components have been examined in the presence of cytosol extracts from a variety of rat tissues. An activity capable of demethylating O6-methyldeoxyguanosine was readily detectable in all tissues examined; arranged in approximate order of decreasing specific activity these are as follows: small intestine, spleen, kidney, lung, liver, skin, heart and brain. The in vitro requirements for the activity derived from liver and the observations that O6-methylguanine and its deoxynucleoside 5'-monophosphate are insensitive to the action of these extracts suggests that this activity may be due to an enzyme which resembles adenosine deaminase. In contrast to the ready degradation of O6-methyldeoxyguanosine the corresponding ethyl derivative was degraded very much more slowly but there was no evidence for other activities against the O4- and O2-methyldeoxythymidines. Similarly, no demethylation of the N-substituted deoxynucleosides, 3-methyldeoxycytidine 3-methldeoxythymidine, 1-methyldeoxyadenosine and 7-methyldeoxyguanosine, was detected.