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Proposes a curriculum to provide all science and mathematics students pursuing careers in secondary teaching with an adequate background to teach a substantial course in physics. Program is based on two years of college level physics and mathematics. (Author/TS)

Summary Amines derived from Brown FK and from its two myotoxic components, 2,4-diamino-5-( p -sulphophenylazo)toluene and 1,3-diamino-4-( p -sulphophenylazo)benzene, were injected intravenously into rats in single doses of 3·13-25 mg/kg. The mixture of amines from Brown FK was also injected into mice in the same range of doses. Cardiac and muscular lesions were produced by the amines in both species. These amines are thought to be the toxic metabolites of Brown FK produced by microbiological degradation in the intestine. The finding that orally administered Brown FK is myotoxic in rats but not in mice is probably due to differences in the intestinal flora in the two species.

Summary Changes in lysosomes associated with hyperoxia were studied in the isolated rat liver, perfused by the method of Mayes and Felts (1966) . Increased PO2 in perfused blood resulted in the formation of enlarged lysosomes and autophagic vacuoles. Demonstration of acid phosphatase (both by light and electron microscopy), β-glucuronidase, aryl sulfatase, and E600-resistant esterase in these organelles identified them as lysosomes. Electron-microscopic observations show that except for changes in the lysosomes, none of the other cellular organelles are distinctly affected. The formation of autophagic vacuoles and the cell damage are prevented if perfusion is carried out with blood oxygenated with air or if the liver is derived from a rat treated with chloroquine. These findings are attributed to alteration in the permeability of the lysosomal membrane resulting from hyperoxia. In turn, this permeability change releases hydrolytic enzymes into the cytoplasm leading to cell death, particularly in the centrilobular areas of the liver in fed and fasted rats. A special role in this process is envisaged for the smaller pericanalicular lysosomes, as indicated by a reduction in their number and a change in their distribution.

The effects of various materials on the chorioallantoic membrane of the 10-day incubated fertile hen's egg have been studied. A detailed sequential histological study of the effects of croton oil and tobacco smoke condensate showed that both substances caused local haemorrhage and necrosis, followed by hyperplasia. The degree of thickening was found to be both dose-and time-dependent. From these observations a test procedure for comparing the irritant activity of tobacco smoke condensates has been developed; this procedure involves measuring the maximum thickness of the membranes 72 h after application of the condensates.

When single daily doses (120 mg/kg) of chloroquine sulphate were given to rats, histochemical and ultrastructural investigations demonstrated a sequence of changes in lysosomes of the liver cell. After the first dose, the liver showed an initial loss of particulate β-glucuronidase and acid phosphatase activities (at 1–3 hours) which was most distinct in the periportal area. By 6–24 hours numerous lysosomes with and without recognizable cytoplasmic organelles (autophagic vacuoles and myeloid bodies, were present. After the second and third doses of chloroquine, the lysosomes increased in size and the majority contained myelin figures. Considerable lysosomal enzyme activity was observed in the periphery of the organelle. Further daily doses produced an increasing amount of undegraded sequestrated material and loss of particulate enzyme staining. There was no demonstrable increase in neutral fat, but a progressive increase in what was probably phospholipid was observed, commencing in those areas of the hepatic lobule showing initial loss of lysosomal enzyme activity. The distributional changes of the phospholipid inclusions correspond closely with those seen for the lysosomal hydrolases. In the highest dosage administered, loss of glycogen occurred in the centrilobular cells, which also showed degenerative changes. We postulate that chloroquine initially labilizes the lysosomal membrane, releasing lysosomal enzymes and initiating autophagy thought responsible for sequenstration of cytoplasmic material. Multiple treatment induces stability of lysosomal membranes and probably impedes entry of enzymes into these organelles, resulting in accumulation of undegraded sequestrated material.

A study has been made of the physicochemical basis of the early local tissue reaction elicited by repeated subcutaneous injections of water-soluble food colourings in the rat. With the exception of Erythrosine BS, the colourings were all absorbed equally rapidly and uniformly, whether from undamaged or from grossly fibrosed subcutaneous tissue. Hence retention at the injection site could not account for the observed differences in local reaction elicited by the colourings investigated. The physicochemical properties found to play a significant part in determining the early local reaction were surface activity, lipid solubility and protein-binding ability, particularly when the latter was associated with alteration of protein structure. No evidence could be found of aggregation of the colouring or of its ability to interact with calf-thymus DNA in vitro . The course of erythrocyte haemolysis and disintegration of cholesterol-lecithin mixed micelles under the influence of increasing concentrations of the sodium salts of Patent Blue V and Blue VRS appeared identical with the corresponding effects brought about by the surfactants sodium lauryl sulphate and triton X-100. Other triphenylmethane colourings possessing considerable surface activity were Light Green SF Yellowish, Fast Green FCF and Brilliant Blue FCF. Most xanthene colourings tested possessed high solubility in tricaprylin and in cholesterol-lecithin mixed micelles. While not displaying surface activity, these colourings affected the integrity of lipid membranes at very low concentrations, in a manner closely similar to that of the amphipathic compounds saponin and lysolecithin. The capacity to bind to protein and, in so doing, to alter protein structure resided in the colourings Light Green SF Yellowish, Fast Green FCF, Brilliant Blue FCF, Erythrosine BS and Indigo Carmine. None of the physicochemical properties described was present to any appreciable extent in the calcium salts of Patent Blue V or Blue VRS, in the xanthene Violamine R or in any azo colouring studied. The physical properties of a colouring were found to correlate closely with type of local reaction elicited by it. In the presence of a marked degree of surface activity, or of lipid solubility, extensive tissue necrosis resulted initially and determined the development of a type IV reaction. Binding with, and alteration of, protein modified the tissue changes by provoking a macrophage response and so leading to a type III reaction. Erythrosine BS was exceptional in that protein binding considerably diminished its lipophilic property, with consequent reduction in the degree of local damage. Colourings possessing none of the physical attributes described produced a mild effect (type I reaction) while a self-limiting reaction (type II) resulted if these properties were present only to a minimal degree. Our investigations lead to the conclusion that the type of the early local tissue reaction produced and, ultimately, the formation of sarcomas by some of the food colourings studied can be fully accounted for on the basis of the physicochemical characteristics of these compounds.

A single intraperitoneal dose of dimethylnitrosamine (DMNA) (30 mg/kg) to rats produces centrilobular hepatocellular necrosis within 18–24 h. Histochemical and electron-microscopic studies of the lysosomal changes occurring during this period show that autophagy and disturbance of lysosomes occur within 35 min of treatment. After 3 h, autophagy is well developed, the majority of cells in the centrilobular area containing a few autophagic vacuoles. These increase in size and number, reaching a peak about 12 h after treatment, when the onset of necrosis is detectable in some cells. The progressive increase in autophagy is not accompanied by a corresponding increase in the number of small lysosomes. Probable mechanisms for this and other observations are discussed.

Summary.— The responses of mouse skin to single (10%) and repeated (1% or 2·5%) applications of sodium lauryl sulphate (SLS), cetrimide and Tween-80 were examined visually and histologically after various periods of exposure. Whereas no damage was caused by Tween-80 when given as single applications or daily for 9 days, SLS and cetrimide both produced damage. After single applications the changes consisted of epidermal cell degeneration, focal necrosis and dermal inflammatory cell infiltration. After repeated treatment acanthosis and hyperkeratosis were identified, and nuclear debris was present in the keratin layer. Cetrimide-treated skins were more severely damaged than those treated with SLS. In general, histological changes preceded visual changes by 1 to 2 days. Irritancy due to these surfactants was related to theri physico-chemical properties. Since Tween-80 at the concentrations used was highly surface active and lipophilic, these properties were not considered of importance in explaining the damage due to SLS or centrimide. Both of these agents react well with protein such as keratin whereas Tween-80 does not. Thus it would seem that this property is of primary importance in the production of damage.

Brown FK was administered to rats at dietary levels of 0.0 (control), 0.001, 0.01, 0.1 and 1.0% for up to 21 wk and to miniature pigs in daily doses of 0, 100, 250 and 500 mg/kg for 24 wk. No effect was observed in either species with respect to growth rate, food consumption, organ weights or liver and kidney function. Apart from the observations described below, no histopathological changes were seen in any organs of the rats or pigs and no significant haematological abnormality was noted in either species. In rats, lipofuscin deposits were observed in the heart, skeletal muscle, kidney tubules, liver parenchymal cells and Kupffer cells at the 1.0% dietary level, in females at 13 wk and in both sexes at 21 wk. At this dietary level, one male showed myocardial changes similar in every respect to those seen following repeated daily administration of Brown FK by stomach tube and reported in paper III of this series (Grasso, Gaunt, Hall, Golberg & Batstone, Fd Cosmet. Toxicol. 1968, 6, 1). After 150 days' treatment, lipofuscin deposits were seen in renal tubular cells also at the 0.01 and 0.1% dietary levels. Deposition of lipofuscin was the principal change observed in pigs. It affected mainly the liver, in which it was observed in both males and females at all dosage levels of Brown FK and was accompanied by increased lysosomal enzyme activity, more marked at the higher dose levels. Lipofuscin was seen also in the heart in males, and here it was associated with an increased acid-phosphatase activity. It was not detectable in the heart in females. Lipofuscin was also present in the kidneys of both sexes, at the highest dose level in females and at all levels in males.

Addition of silver acetate to the diet (130–1000 ppm) or drinking water (1500 ppm) of weanling rats fed a vitamin E-deficient diet precipitates a rapidly fatal liver necrosis on Day 14 or subsequently. In the prenecrotic stage (11–13 days), the hepatocellular changes were characterized by nuclear vacuolation, by an increase in the number and size of lysosomes and by their movement away from the peribiliary area. The mitochondria were enlarged and their shape varied considerably. Some mitochondria contained rough granular material, while in others the number of electron-dense granules was reduced. Hepatocellular necrosis appearing initially in the centrilobular area rapidly involved the whole lobule. Degeneration of nuclei, mitochondria, and endoplasmic reticulum seemed to occur simultaneously. After an interval, macrophages infiltrated the necrotic area. Numerous large lysosomes containing electron-dense material (probably containing silver) could be demonstrated in these cells. No changes were observed in livers of rats given silver acetate and vitamin-E supplements. The mitochondrial changes possessed some of the features seen in rats fed a torula-yeast diet deficient in vitamin E and selenium, suggesting some degree of dysfunction. The lysosomal changes may be a consequence of such possible dysfunction. To account for the interrelationships between vitamin E, selenium, and silver seen in these and other experiments, a mechanism is put forward whereby, in the absence of vitamin E, silver reduces the availability of selenium as “selenide” in nonheme iron proteins. The result would be an impaired ability of these proteins to function as electron carriers. The widespread distribution of these proteins in nucleus, mitochondria, and endoplasmic reticulum could account for the contemporaneous lysis seen in these organelles.

Four samples of PVC containing 0, 0·17, 0·5 and 1·4% dibutyltin diacetate but otherwise identical in every respect, were used to evaluate two tissue culture methods frequently used in the safety testing of plastics. In one method (agar overlay technique), primary neonatal rat-kidney cells were grown in petri dishes, covered with 1% agar and stained with neutral red. Pieces of the plastics to be tested were then placed on the agar and the plates were examined 24 hr later. The area of neutral-red loss underneath each piece of PVC was taken as an index of toxicity. In the other method, primary neonatal rat-kidney cells were maintained in a growth medium made with serum previously used to extract dibutyltin diacetate from the plastics. In this case, lysosomal acid-phosphatase activity was used in addition to loss of neutral red to indicate cytotoxicity. With both methods, the extent of cell necrosis was in direct proportion to the concentration of dibutyltin diacetate in the plastics. The agar overlay method proved to be the more sensitive in detecting low concentrations of the cytotoxic agent in the plastics. The histochemical method demonstrated cytotoxic changes in the cells much earlier than did the loss of neutral red, but did not improve the sensitivity of the serum extract technique.

Acute oral toxicity studies with Brown FK gave LD50 values greater than 2 g/kg and 8 g/kg in mice and rats respectively. The values for intraperitoneal injection were 1·5–2 g/kg in mice and 0·75–1·15 g/kg in rats. When repeated daily oral doses ranging from 0·1 to 2 g/kg were given to rats, a precipitate weight loss and a vacuolar myopathy of the heart and skeletal muscles accompanied by lipofuscin formation occurred with doses of 0·5 g/kg and over. At the highest dose employed (2 g/kg) there was also hepatic centrilobular necrosis and renal tubular degeneration. Pretreatment with antibiotics reduced the incidence of muscle lesions. Repeated intraperitoneal injection did not have any effect on the heart or skeletal muscle. Only two of the six components of Brown FK, compound I (2,4-diamino-5-(p-sulphophenylazo)toluene) and compound II (1,3-diamino-4-(p-sulphophenylazo)benzene), were found to produce muscle damage after repeated oral doses of 0·5 g/kg. Comparable doses of mixtures containing the other components as well as I or II were considerably less toxic. After daily oral doses of 1 g/kg, muscle lesions were found in rats, rabbits and guinea-pigs, but none were seen in mice or hamsters. The results suggest that the intestinal microflora is responsible for the breakdown of the components of Brown FK to toxic products. Variations between species and between animals of the same species may be due to qualitative and quantitative differences in the intestinal microbial population.

Acute toxicity studies in rats and mice and a short-term study in rats have been carried out on Sunset Yellow FCF. The acute intraperitoneal LD50 ranged from 3·8 to 5·5 g/kg in rats and mice. Orally, doses up to 10 g/kg in rats and 6 g/kg in mice were tolerated without ill effect. Feeding of Sunset Yellow FCF to rats at dietary levels of 0·0 (control), 0·5, 1·0, 2·0 or 3·0% for 90 days evoked no adverse effect on growth or food consumption but there was slight diarrhoea throughout the study at 3% and during the first few weeks at 2%. There was no departure from normality in the haematological investigations or in the terminal liver and kidney function tests. At autopsy, the caecum was enlarged at the 2 and 3% levels and the testes at the 3% level. There were no histological changes attributable to Sunset Yellow FCF. A no-effect level of 1% Sunset Yellow FCF was established in the diet for 90 days, a level equivalent to 500 mg/kg/day.

A study has been made of the local lesions produced in rats (and in one instance in mice) by a short series of subcutaneous injections of food colourings belonging to various chemical categories. The resulting pathological changes have been separated into four main categories. In the type I response (Patent Blue V and Amaranth in rats and Blue VRS in mice) no appreciable connective tissue reaction was produced, while in the type II response (Erythrosine and Green S in rats) the connective tissue reaction developed to a limited degree. The nature of these responses suggested an inability of the test material to produce injury in the normal subcutaneous tissue or to interfere with the process of connective tissue repair whenever the normal integument became necrotic. These responses were produced by colourings which failed to produce sarcoma when injected repeatedly at the same site for 10 months or longer. Type III response (Light Green SF Yellowish, Brilliant Blue FCF and Fast Green FCF in rats) consisted mainly of an intense macrophage response involving macrophage necrosis and considerable fibroblastic proliferation. This effect closely resembles the tissue reaction reported by other workers following repeated injections of high doses of iron-dextran and of carboxymethylcellulose. Type IV response (Blue VRS and Eosine G in rats) resulted in the development of thick collagenous tissue with foci of persistent fibroblastic reaction. This lesion was found to correspond with the previously reported early stages of the fibrous reaction around subcutaneous implants of films of solid material. Colourings that have been reported by previous authors to induce subcutaneous sarcoma at the site of repeated injection produced either a type III or a type IV response. Thus a close correlation has been found to exist between the category of initial tissue response and the long-term outcome of repeated injections, in terms of the development of subcutaneous sarcoma at the site of injection. These results indicate the possibility that sarcoma production by the colourings under consideration results from derangement of connective tissue repair rather than from induction by a process of chemical carcinogenesis.

After acute oral chloroquine administration in rats, there was an accumulation of membrane whorls (myeloid bodies) in certain cell organelles of the heart. Acid phosphatase staining indicated that these were lysosomal in nature. With up to six daily doses of chloroquine (120 mg/kg), the myeloid bodies increased in size and number. One week after the drug treatment was discontinued, the myeloid bodies were smaller and showed increased acid phosphatase activity. After a further 2 weeks they had disappeared, showing that the effect of the drug was reversible.

A short-term feeding study has been carried out on lauric diethanolamide (LDE) in the rat. Dietary levels of 0.0 (control), 0.1, 0.5, 1.0 and 2.0% for 90 days evoked no adverse effect on the appearance or condition of the animals. Growth retardation was associated with diminished food intake, at and above the 0.5% level. Food refusal was demonstrably due to an effect of the test material on palatability of the diet. Terminal haematological examination revealed a reduction in the haemoglobin level, haematocrit and red cell count at the 1 and 2% levels in females but less pronounced effects were seen in males. Serum levels of glutamic-oxaloacetic transaminase were elevated at dietary levels of 0.5% and above in females but only at 0.5% in males. No untoward effect was observed in the renal function tests. The principal organ weight changes were: increases in the relative kidney weight in all test groups except at 0.1% in females and at 0.1 and 0.5% in males; and increases in the relative liver weight in females on the two highest levels. The types and incidence of histological lesions were comparable in control and test groups. A no-effect level was established of 0.1% LDE in the diet of rats for 90 days, a level equivalent to 50 mg/kg/day.

THE histochemical detection of early changes in the characteristics, distribution and enzyme contents of lysosomes, as well as their cycles of appearance and disappearance, affords a sensitive index of toxic effects on the cell. Hypoxia is known to labilize lysosomes1 and might be expected to render them more susceptible to adverse influences. Selective uptake of neutral red by lysosomes2,3 and the stabilization brought about by chloroquine4 constitute further experimental manipulations to which lysosomes can be subjected. We have made detailed observations of lysosomal behaviour in these conditions as a preliminary to the study of lysosomal changes in the damaged liver of the rat.

nachweis, dass hohe Strontiumkonzentrationen die Kalzifikation der, Knochen neugeborener Mause, und diejenige in Knochengewebskulturen hemmen. Im Gegensatz dazu und zu anderen knochenaffinen metallen wirkt Strontiumnitrat in Dosen von25, 50, 100 and 200 mg/kg, verabreicht wahrend des 9. bis 19. Trachtigkeitstages, bei Ratten nicht teratogen.