The myofilament arrangement in the femoral muscle of the cockroach, Leucophaea maderae fabricius.

The structure of the femoral muscle of the cockroach, Leucophaea maderae, was investigated by light and electron microscopy. The several hundred fibers of either the extensor or flexor muscle are 20 to 40 micro in diameter in transverse sections and are subdivided into closely packed myofibrils. In glutaraldehyde-fixed and epoxy resin-embedded material of stretched fibers, the A band is about 4.5 micro long, the thin filaments are about 2.3 micro in length, the H zone and I band vary with the amount of stretch, and the M band is absent. The transverse sections of the filaments reveal in the area of a single overlap of thick and thin filaments an array of 10 to 12 thin filaments encircling each thick filament; whereas, in the area of double overlap in which the thin filaments interdigitate from opposite ends of the A band, the thin filaments show a twofold increase in number. The thick filament is approximately 205 to 185 A in diameter along most of its length, but at about 0.2 micro from the end it tapers to a point. Furthermore, some well oriented, very thin transverse sections show these filaments to have electron-transparent cores. The diameter of the thin filament is about 70 A. Transverse sections exhibit the sarcolemma invaginating clearly at regular intervals into the lateral regions of the A band. Three distinct types of mitochondria are associated with the muscle: an oval, an elongate, and a type with three processes. It is evident, in this muscle, that the sliding filament hypothesis is valid, and that perhaps the function of the extra thin filaments is to increase the tensile strength of the fiber and to create additional reactive sites between the thick and thin filaments. These sites are probably required for the functioning of the long sarcomeres.

The filament lattice of cockroach thoracic muscle.

The fine structure of the tergo-coxal muscle of the cockroach, Leucophaea maderae, has been studied with the electron microscope. This muscle differs from some other types of insect flight muscles inasmuch as the ratio of thin to thick filaments is 4 instead of the characteristic 3. The cockroach flight muscle also differs from the cockroach femoral muscle in thin to thick filament ratios and diameters and in lengths of thick filaments. A comparison of these latter three parameters in a number of vertebrate and invertebrate muscles suggests in general that the diameters and lengths of the thick filaments and thin to thick filament ratios are related.

The sarcoplasmic reticulum and its association with the T system in an insect.

The fine structure of the sarcoplasmic reticulum and the transverse tubular system of the femoral muscle of the cockroach, Leucophaea maderae, was studied after prefixation in glutaraldehyde, postfixation in osmium tetroxide, and embedding in Epon. The sarcoplasmic reticulum in this muscle reveals features not previously reported. The sarcoplasmic reticulum is abundant, consisting mainly of a fenestrated envelope which surrounds each myofibril at all levels in the sarcomere. This sarcoplasmic reticulum envelope is continuous transversally as well as longitudinally along the myofibrils. Dyadic junctions are formed by a single T system element which contacts the unfenestrated sarcoplasmic reticulum of adjacent myofibrils in an alternating manner at the ends of the A band. At the dyads, regularly spaced thickenings of the sarcoplasmic reticulum membranes bordering the dyadic spaces are noted. These thickenings, however, do not contact the T tubule membrane. Typical dyadic contacts also are seen between the cell surface membrane and sarcoplasmic reticulum. Z line-like material is seen in contact with the membranes of the cell surface and longitudinal branches of the T systems.

An electron microscope autoradiographic study of the neuronal and extraneuronal localization of labeled amine in the heart of the bat after administration of tritiated norepinephrine.

The localization of labeled amine in the heart of the bat after administration of tritiated norepinephrine (NE) was studied by means of electron microscope autoradiography. Monoamine oxidase was inhibited so that the distribution of amine in both neuronal (Uptake(1)) and extraneuronal (Uptake(2)) sites could be analyzed. Labeling was nonrandom in both the atrial and ventricular myocardium. The highest relative specific activity was found in neural processes which showed morphological criteria of terminal adrenergic axons. Analysis of the distribution of label around the labeled axonal varicosities indicated that the radioactive amine was more concentrated peripherally than centrally in these structures. Label was also found over cardiocytes in both atrium and ventricle. The pattern of this labeling indicated that the radioactive amine was associated with myofilaments. In the ventricle, I bands were most heavily labeled, indicating a probable association of radioactive amine with thin filaments. Labeling was prevented by administration of phenoxybenzamine and decreased only in cardiocytes by normetanephrine. The nonrandom distribution of labeled amine within cardiocytes supports the view that Uptake(2) represents not only a second mechanism of inactivation of the sympathetic neurotransmitter, but may also be involved in the mediation of some of the action of NE on cardiac muscle.

The uptake and secretion of 3-methylcholanthrene by the prostate glands of the rat and dog.

In anesthetized rats in which prostatic fluid was collected over a 2-hour interval from 24 to 26 hours after a single ip dose of 5 mg [6-14C]3-methylcholanthrene ([14C]MCA)/kg, radioactivity was found in the fluid at levels only slightly less than those in plasma; at 26 hours after treatment, levels of radioactivity within the prostate were higher than those in prostatic fluid or plasma. When unanesthetized dogs with surgically prepared prostatic fistulas were given a single ip dose of 0.5 mg [14C]MCA/kg and when serial prostatic fluid and plasma samples were collected over the ensuing 50 hours (2 dogs) or 212 hours (1 dog), radioactivity appeared in the prostatic fluid at levels initially greater than those in plasma and then fell progressively with time to less than those of plasma. At 50 hours after treatment, radioactivity was recovered from the prostate glands of 2 dogs with fistula and 2 dogs without fistula at levels of about one-fourth those of plasma. Thus it was found that [14C]MCA and/or its metabolites entered the prostate glands and prostatic fluids of the rat and dog.

Detection of sulfur mustard-induced DNA modifications.

Sulfur mustard is acutely toxic to the skin, eyes, and respiratory tract, and is considered carcinogenic to humans by the IARC. Since all of these toxicities are thought to be initiated by DNA alkylation, the level of DNA damage should serve as a biomarker for exposure. To develop methods of detecting this damage, DNA was modified by [14C]-labeled sulfur mustard and DNA adducts were released by mild acid hydrolysis. Radioactivity co-eluted on HPLC analysis with marker 7-(2-hydroxyethylthioethyl) guanine and 3-(2-hydroxyethylthio-ethyl) adenine synthesized from 2-chloroethyl 2-hydroxy-ethyl sulfide. Unambiguous identification of the major adduct, 7-(2-hydroxy-ethylthioethyl) guanine, was provided by gas chromatography combined with mass spectrometric detection. The most abundant adduct, 7-(2-hydroxyethyl-thioethyl) guanine, accounted for 61% of the total alkylation and could be detected as a fluorescent HPLC peak with a detection limit of 10 pmol. To demonstrate the applicability of this method to biological samples, DNA was extracted from the white blood cells of human blood exposed to 131 microM sulfur mustard in vitro and shown to contain 470 pmol of 7-(2-hydroxyethylthio-ethyl) guanine per mg of DNA.

Uptake and secretion of carcinogenic chemicals by the dog and rat prostate.

Because the prostate of laboratory animals seems relatively resistant to the carcinogenic effects of systematically administered chemicals--an observation of some significance in attempts to establish the etiology of human prostatic adenocarcinoma and to produce animal models of prostatic cancer--we studied the penetration of eight carcinogenic chemicals into both the prostate gland and the prostatic secretion of the dog and the rat. The eight chemicals were 3-methylcholanthrene, 7,12-dimethylbenz[a]anthracene, N-hydroxyurethane, aflatoxin B1, 3-amino-1,2,4-triazole, 2-acetylaminofluorene, N-Methyl-N'-nitro-N-nitrosoguanidine, and cadmium. In both species all eight substances and/or their metabolites were found to rapidly enter the prostate, and all except cadmium were recovered from prostatic fluid. Thus, although the levels in prostatic fluid did not always reflect levels in the gland, there was little if any barrier to the penetration of these chemicals into the gland. The apparent relative refractoriness of the prostate to systematically administered carcinogenic chemicals cannot be due to failure of such substances to enter the gland.

The microscopic distribution of tetracycline in human teeth.

The distribution of tetracycline fluorescence was studied in a random collection of 378 teeth extracted in 1976 in Farmington, Connecticut, using ultra-violet microscopy of longitudinal ground sections. The sample consisted of 4 deciduous molars and the following numbers of permanent teeth: 72 incisors, 41 canines, 63 premolars, 26 first molars, 24 second molars, and 148 third molars. Narrow fluorescent lines reflecting short regimens of drug administration and broad bands corresponding to long term administration were identified in the dentin and tabulated in relation to their anatomical position. Fluorescence in the cementum was also noted. Of all the teeth, 33.3% exhibited tetracycline changes in either the dentin, the cementum, or both. There were 89 affected third molars. These had a mean of 3.1 (+/- 2.9) fluorescent dentin lines per tooth as well as many broad bands, largely in the root region. The teeth were subjected to an estimation of age in order to segregate those teeth whose dentin had formed prior to the advent of tetracycline usage. When this was done, the corrected incidence of tetracycline affected teeth in the collection was 45.4%. The observations show that American teeth have been extensively affected by tetracycline. The location of fluorescent markings appear to reflect reluctance to use this drug during the developmental period when cosmetic effects could result. Markings clearly indicate heavy usage of tetracycline during a later period of development corresponding to third molar root formation.

Lipid droplet accumulation in cardiac muscle cells of the bat: potential auto-toxicity of the cardiac sympathetic innervation.

The previously described ability of reserpine and parachlorophenylalanine to induce the accumulation of lipid droplets in ventricular cardiac muscle cells of the bat was investigated. Lipid droplet accumulation was assessed qualitatively by light microscopy and quantitatively by morphometric analysis of electron micrographs. An hypothesis that the action of the drugs was an indirect one, mediated by the cardiac adrenergic innervation, was framed and tested. Lipid droplet accumulation occurred during a time of intense sympathetic activity, that of arousal from hibernation. The ability of the two drugs to produce the effect was antagonized by prior sympathetectomy with 6-hydroxy-dopamine. The effect was mimicked by administration of exogenous norepinephrine together with inhibitors of its catabolic enzymes, monoamine oxidase and catechol-o-methyl transferase. These observations are all consistent with the initial hypothesis and raise the possibility that endogenous norepinephrine in the cardiac sympathetic innervation might be, at least potentially, auto-toxic.

Carcinogenicity of 4-chloro-o-phenylenediamine, 4-chloro-m-phenylenediamine, and 2-chloro-p-phenylenediamine in Fischer 344 rats and B6C3F1 mice.

In both male and female Fischer rats, feeding 4-chloro-o-phenylenediamine at 0.5 or 1% in the diet led to a significant increase in hyperplasia, papilloma and carcinoma of the urinary bladder. Neoplastic nodules of the liver and squamous cell papillomas of the stomach were also increased slight. Similar levels of this compound in male and female B6C3F1 mice increased significantly the incidence of hepatocellular adenomas and carcinomas. 4-Chloro-m-phenylenediamine at 0.2 or 0.4% in the diet led to a significant elevation in the incidence of adrenal pheochromocytoma in male rats. At 1 or 2% levels it caused hepatocellular adenoma in female mice. Although the analog, 2-chloro-p-phenylenediamine increased transitional cell hyperplasia of the kidney in both male and female rats, it had no significant neoplastic effect at the 0.15 or 0.3% levels fed.

Hemangioendothelial sarcoma of penis.

Hemangioendothelial sarcoma of the penis in a 44-year-old man was treated by preoperative radiation, penectomy, and chemotherapy. The patient was free of evident disease 2 years later. Electronmicroscopy showed differentiated vascular structures at the periphery of the lesion and anaplastic cells throughout the remainder of the tumor.

Thirteen-week toxicology studies of 1-amino-2,4-dibromoanthraquinone in Fischer 344/N rats and B6C3F1 mice.

1-Amino-2,4-dibromoanthraquinone (ADBAQ), an intermediate in the production of commercial dyes for wool, silk, and synthetic fibers, was selected for toxicology and carcinogenesis studies in two rodent species. In advance of the 2-year studies, 13-week studies were conducted in male and female F344/N rats and B6C3F1 mice which were fed a diet containing ADBAQ at concentrations of 0, 0.25, 0.50, 1.00, 2.50, and 5.00%. ADBAQ stained the skin and fur red at all doses in rats and at 1.00% and higher concentrations in mice. Lethargy and emaciation were noted at the 2.50% and higher doses in rats of both sexes. In general, the absolute weight of the liver and the liver/organ weight ratios increased in both sexes and species at all doses. Treated rats developed a chronic toxic hepatitis characterized by hepatocytomegaly, centrilobular vacuolar degeneration and necrosis, regenerative nodules, acute necrotizing cholangitis, bile duct hyperplasia, chronic active inflammation in periportal areas, and focal pigmentation. The hepatopathy occurred at all doses in males and at 0.50% and higher in females and correlated with elevations of serum glutamic-pyruvic and glutamic-oxaloacetic transaminases, leukocytosis, and neutrophilia. Hyaline droplet degeneration in the proximal convoluted tubules of the kidneys occurred in male rats, and uterine atrophy was observed in female rats at 1.00% and higher. Anemia occurred in both sexes of rats at all doses and thymic atrophy was observed in both sexes of high-dose rats. In male mice minimal dose-related lesions in the liver included centrilobular glycogen depletion at 1.00% and higher and pigmentation at all doses. At comparable doses, ADBAQ was considered to be markedly toxic in rats and of minimal nonlife-threatening toxicity in mice.

Analysis of chlorthalidone in biological fluids by high-performance liquid chromatography using a rapid column cleanup procedure.

A high-performance liquid chromatographic assay usable for clinical monitoring of chlorthalidone in biological fluids was developed. Extraction efficiency was greater than 80% for blood and urine using a rapid, disposable column cleanup procedure. Chlorthalidone could be reliably measured in the range of 100-4,000 ng/ml in biological fluids with excellent day-to-day reproducibility and within-day precision. Chlorthalidone was found to be stable at -20 degrees C in blood and urine for at least 1 year, permitting repeat assays and large clinical studies to be conducted. The pharmacokinetics of chlorthalidone was studied in 24 subjects over a 120-h time interval following a single dose. chlorthalidone has a long terminal half-life in whole blood of 49 h, with peak concentrations occurring 8-10 h after oral dosing. During the first 12 h after dosing, chlorthalidone was rapidly excreted into urine followed by a slower phase with a half-life of 49 h.