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In rats, 1-nitronaphthalene (1-NN) causes both pulmonary and hepatic toxicity. Pulmonary toxicity is evident as bronchiolar damage, with necrosis of Clara cells and ciliated cells, whereas hepatic injury involves vacuolation of centrilobular hepatocytes. Pretreatment with O,O,S-trimethylphosphorodithioate [OOS-MeP(S)] or p-xylene gave three- to fourfold protection against 1-NN toxicity. These pretreatments also prevented both the increase in lung weight and the rise in gamma-glutamyltranspeptidase and alkaline phosphatase activity in bronchoalveolar lavage fluid normally associated with 1-NN toxicity. Pretreatment with Aroclor 1254 or beta-naphthoflavone (beta-NF) did not alter the LD50 of 1-NN. Aroclor or beta-NF pretreatment did, however, prevent morphological signs of lung injury and any increase in either lung weight or enzyme activity in bronchoalveolar lavage fluid. Liver damage was not prevented by these treatments; indeed, injury was exacerbated and was transferred from centrilobular to periportal areas. In control rats the covalent binding of [1-14C]NN to liver microsomes was eight times greater than to lung microsomes. Pretreatment with OOS-MeP(S) decreased covalent binding to lung microsomes, without affecting binding to liver microsomes. Conversely, both Aroclor and beta-NF slightly increased covalent binding in lung, but increased liver binding by 250-300%. Phenobarbitone also increased binding to liver microsomes by 250-300%, but failed to increase, or alter, the distribution of liver damage. The reported effects of these pretreatments indicate that the toxicity of 1-NN is probably activated by isoenzyme CYP2B1 in lung, but by isoenzymes CYP1A1 or CYP1A2 in the liver.

A novel procedure for the isolation of Clara cells from rat lung is described. Single-cell suspensions from male F344/TOX rat lungs, prepared by subtilisin digestion, were treated with monochlorobimane (10 microM) and anlaysed by fluorescence-activated flow cytometry. A sub-population of about 2.8% of the total, showing the highest blue fluorescence and by inference the highest GSH concentration, was isolated as Clara cells of > 95% purity. Type II cells of similar purity were sorted after treatment with Phosphine 3R. Both sub-populations were > 90% viable, as judged by Trypan Blue exclusion. Comparison of CYP (cytochrome P-450) isoenzymes between these subpopulations, using Western blotting, showed CYP1A1 to be barely detectable. In Clara cells, CYP2B1 was 10-fold higher than in Type II cells. Mono-oxygenase activity towards the O-deethylation of 3-cyano-7-ethoxycoumarin was 2-fold higher in Clara cells. No activity was detected in macrophages. Pretreating rats with the mono-oxygenase inducers phenobarbitone, 3-methylcholanthrene or Aroclor 1254 showed the last-named to be the most potent inducer of CYP1A1. In Clara cells, CYP1A1 concentration and mono-oxygenase activities were induced > 2000- and 50-fold respectively, whereas in Type II cells increases of 300- and 3.6-fold were seen. Clara cells isolated from the lungs of control rats had a concentration of GSH (2.7 nmol/10(6) cells) that was 9- and 2-fold higher than that in Type II cells or macrophages respectively. GSH depleted by monochlorobimane treatment was restored after 2-3 h incubation with 0.2 M N-acetylcysteine. gamma-Glutamyltranspeptidase activity in Clara cells was 6- and 50-fold higher than in Type II cells or macrophages respectively.

Apoptosis, a major form of cell death in the immune system, is characterized by cell shrinkage, chromatin condensation, and cleavage into nucleosomal fragments. Apoptosis may be the mechanism for the elimination of autoreactive and unselected CD4+ CD8+ thymocytes in the thymus. A large number of diverse agents are capable of inducing apoptosis in immature thymocytes. Rat thymocytes were treated with etoposide, a DNA topoisomerase II reactive agent, or dexamethasone, a glucocorticoid, and separated on discontinuous Percoll gradients. We have identified and isolated a transitional preapoptotic population of thymocytes that exhibited early morphologic and biochemical changes associated with apoptosis. These preapoptotic cells were intermediate in size and density between normal and apoptotic thymocytes and exhibited a decreased surface expression of both CD4 and CD8 molecules compared to control thymocytes. On ultrastructural examination, they were shown to possess sharply defined clumps of condensed chromatin abutting onto the nuclear membrane. These morphologic changes, the first detectable signs of apoptosis, occurred prior to the internucleosomal cleavage of DNA, often regarded as the biochemical hallmark of apoptosis. Nucleosomal fragments of 180 to 200 base pairs or multiples thereof were, however, detected following subsequent dramatic changes in the nuclear structure of these preapoptotic cells that resulted in morphology typical of apoptosis. These results suggest that early critical events in apoptosis precede internucleosomal cleavage of DNA.

The lungs of rats exposed to formaldehyde vapor, for 6 hr/day over 4 consecutive days, were examined for signs of injury and for changes in the level, or activity, of cytochrome P450. The animals were supplied with 10 ppm formaldehyde vapor generated, in two separate experiments, either from an aqueous solution of formaldehyde or from heated paraformaldehyde. All rats were exposed for 6 hr, on each of 4 consecutive days, and killed 1 day after the onset of the fourth period of exposure. The lung weights and gains in body weight of exposed animals were indistinguishable from those of their controls. Lungs from the formaldehyde-exposed animals did not show any signs of injury, even at the ultrastructural level. Bronchoalveolar lavage samples from exposed animals showed no increase in alkaline phosphatase or gamma-glutamyl transpeptidase activity. The total concentration of cytochrome P450 in the lungs of exposed animals was similar to that found in their controls. The P450 activity of pulmonary microsomes from exposed animals was not significantly different from that obtained with samples from the control animals. These results indicate that repeated exposure to 10 ppm formaldehyde vapor does not injure the deep lung of rats and has no effect on the level of lung P450 or on its activity against substrates for the most common pulmonary forms of this enzyme.

Apoptosis, a major form of cell death, is characterized by chromatin condensation, a reduction in cell volume and endonuclease cleavage of DNA into oligonucleosomal length fragments. The detection of these fragments by gel electrophoresis, as a DNA ladder, is currently used as the major biochemical index of apoptosis. Here we report that key morphological changes of apoptosis can be dissociated experimentally from the DNA fragmentation produced by endonuclease activity. Internucleosomal cleavage of DNA is thus likely to be a later event in the apoptotic process.

Using flow cytometry, we describe a method for separating and quantifying normal and apoptotic thymocytes. Apoptosis was induced in isolated thymocytes from immature rats by treatment with the glucocorticoid dexamethasone or the antitumor agent etoposide. Subsequent incubation with the vital bisbenzimidazole dye Hoechst 33342 and the DNA intercalating agent propidium iodide enabled three distinct populations of cells to be identified and sorted by flow cytometry. Dead cells fluoresced red due to propidium iodide whereas normal and apoptotic cells fluoresced blue due to Hoechst 33342. Apoptotic cells were distinguished from normal thymocytes both by their higher intensity of blue fluorescence and by their smaller size as determined by a reduction in forward light scatter. The larger cells, with low blue fluorescence, showed normal thymocyte morphology by electron microscopy and the absence of any DNA fragmentation as measured by agarose gel electrophoresis. In contrast, the smaller cells showed both the morphological characteristics of apoptosis and extensive internucleosomal fragmentation of DNA to multiples of approximately 180 bp. Using this method, a time-dependent induction of apoptosis by dexamethasone, which was inhibited by cycloheximide, actinomycin D, and aurin tricarboxylate, was observed. The method should facilitate mechanistic studies on the induction of apoptosis in thymocytes.

gamma-Glutamyltranspeptidase (gamma-GT) was localized in slices of rat lung, at the ultrastructural level, by pre-embedding immunogold labelling. Antiserum was raised against the protein purified from rat kidney. The enzyme was found to be concentrated on the lumenal surface of the non-ciliated ("Clara") cells of the bronchiolar epithelium and, to a lesser degree, on the surface of type II alveolar pneumocytes. This immunological localization was consistent with the distribution of reaction product, in both slices and resin sections incubated to demonstrate gamma-GT activity. gamma-GT is probably involved in the utilization of reduced glutathione (GSH) present in the fluid lining the airway epithelium.

After a single subcutaneous dose of iron-dextran (600 mg of iron/kg), iron overload developed in C57BL/10ScSn mice. At 4, 24 and 78 wk liver nonheme iron concentrations were 67-, 42- and 21-fold higher than controls, respectively. Much of the iron was in macrophages, but hepatocytes were also strongly positive for Perls' stainable iron. One feature was the development of iron-positive nuclear inclusions in hepatocytes. After a delay of at least 8 wk when no stainable iron was evident, a maximum of 37% of periportal hepatocytes contained inclusions by 24 wk. Although this proportion remained constant for the remainder of the study, the size of the inclusions (which were not membrane-limited) increased to greater than 3 microns in diameter, occupying greater than 25% of the nuclear volume. The presence of iron in the inclusions was confirmed by energy dispersive x-ray microanalysis. Immunocytochemical studies showed that the iron was present as aggregates of ferritin. Quantitation of nonaggregated ferritin molecules by image analyses after electron microscopy demonstrated that within 4 wk ferritin levels in cytoplasm and nucleoplasm had greatly increased but that there was a concentration gradient of approximately one order of magnitude across the nuclear envelope. These findings are consistent with the hypothesis that in iron-loaded mouse hepatocytes there is a slow passage of ferritin-molecules through the nuclear pores; the gradient is maintained by the continual aggregation of ferritin within the nucleus. Intranuclear ferritin may provide a source of iron for catalyzing hydroxyl radical formation in nuclei during some toxic, carcinogenic and aging processes.

Cellular changes occurring in mouse lung following the administration of myomontanone (MM) were compared with those due to another 3-substituted furan compound, 3-(N-ethylcarbamoyloxymethyl)furan (ECMF). The i.p. administration of lethal doses of these furans resulted in two different forms of lung injury. ECMF was particularly oedemagenic; it resulted in early damage to endothelial cells lining the alveolar capillaries and it also damaged the Clara cells of the terminal bronchioli. Myomontanone was much less oedemagenic and, initially, resulted in minimal cell damage. These minor changes were, however, followed by a delayed but acute injury to the type I pneumocytes, which progressed to elicit the proliferation of type II cells. These two forms of injury probably reflect significant differences in the mechanism of toxicity of the two compounds. The changes following dosing with ECMF resemble those reported after the administration of many other furans, e.g. 4-ipomeanol. The selective injury to type I pneumocytes, observed after MM, has been reported after the administration of many, apparently unrelated, compounds. The late development of MM-induced injury, long after the compound has presumably been cleared from the circulation, may result from the release of reactive metabolites which had previously been bound, reversibly, to cellular macromolecules.

Interstitial lung disease was diagnosed in a 52-yr-old male diamond polisher, who worked with polishing disks containing cobalt. After a further 7 months of probably high occupational exposure without any specific treatment, he had to quit work because of dyspnea. Despite treatment with systemic corticosteroids and continuous oxygen administration, he died 3 months later in respiratory distress. Postmortem examination of the lung tissue showed a typical giant-cell interstitial fibrosis, with active inflammatory cell infiltration superimposed on an established centrilobular fibrosis. The lung tissue contained 2.1 micrograms cobalt/g wet weight (more than 100-fold the normal concentration); cobalt particles, mainly localized in macrophages, were identified by transmission electron microscopy and energy-dispersive X-ray analysis. We speculate that the rapid deterioration and fatal outcome resulted from the continued exposure to cobalt, leading to a high pulmonary concentration of cobalt, and from the oxygen treatment because cobalt promotes the formation of hydroxyl free radicals.

Protection afforded by trialkyl phosphorothionates against the lung injury caused by trialkyl phosphorothiolates probably results from the inhibition by the P = S moiety of the thionates, of one or more pulmonary cytochrome P-450 isozymes. The aromatic hydrocarbons p-xylene and pseudocumene also protect against this injury and inhibit some P-450 isozymes, but by a different mechanism. OOS-Trimethylphosphorothionate and p-xylene were compared as protective agents against the effect of OOS-trimethylphosphorothiolate and two other lung toxins ipomeanol and 1-nitronaphthalene that are known to be activated by cytochrome P-450. The effects of these protective compounds, in vivo, on pulmonary cytochrome P-450 activity were also determined. Both compounds inhibited pentoxyresorufin O-deethylase activity, but not ethoxyresorufin O-deethylase. The phosphorothionate was most effective against lung injury caused by the phosphorothiolates and 1-nitronaphthalene, whereas p-xylene was much more effective against ipomeanol. beta-Naphthoflavone, which induces pulmonary ethoxyresorufin O-deethylase activity, did not protect against phosphorothiolate or 1-nitronaphthalene injury, and it was only marginally effective in decreasing the toxicity of ipomeanol.

The intestine of lambs killed immediately after birth and at intervals after the first feed was studied by electron microscope cytochemistry. Ferritin, incorporated into this feed, was found within 2 h of feeding within vesicles throughout the cytoplasm of enterocytes lining the proximal and mid-intestine. Some of these vesicles had fused with the lateral and basal membranes of the enterocytes. Histochemical reaction products for alkaline phosphatase and a series of lysosomal enzymes were localized within the vesicles; the distribution of acid hydrolases, however, was not uniform within each cell. Biochemical estimations of the activity of these enzymes showed greatest activity in the distal intestine of the newborn lamb. The activity of only one of these enzymes, N-acetyl-beta-glucosaminidase, was maximal in the mid-intestine. These observations indicate that cytoplasmic vesicles, translocating proteins across the enterocyte, probably carry intestinal alkaline phosphatase activity in their limiting membrane. Lysosomal enzymes, particularly glucosaminidase, are introduced into these vesicles as they traverse the enterocytes of the mid-intestine. A less specialized complement of lysosomal enzymes is probably introduced into vesicles in the distal intestine where ingested protein may be digested, rather than transported across the cell.

Paneth cells containing zinc-rich granules were found in the small intestine of 6-day-old rats. These cells were more numerous in older animals and were consistently most common in the distal ileum. The zinc content of granules from 10-day-old rats was similar to that found in adults (ca 300 mg atoms/kg dry weight) but no calcium could be detected. An injection of cortisone acetate at 5 days resulted in a premature increase in the numbers of Paneth cells in 10-day-old rats. The cell granules contained normal, adult levels of zinc, a calcium concentration of ca 400 mg atoms/kg dry weight and also an increased concentration of phosphorus.

In the liver of pregnant rats, fed ad libitum, there was an increase in acid phosphatase specific activity which occurred in two peaks, one at the 15th day and the other at the end of gestation. By light and electron microscopic histochemistry, the activity was found to be localized in parenchymal cell peribiliary dense bodies and also in phagosomes present in macrophages and parenchymal cells. There was an increase in liver wieght which reached a peak at the 17th day of gestation. Total DNA also rose to the 17th day; there was a high rate of cell division in the hepatic parenchyma at the 17th and 18th days of gestation. During this period single cell deletion by apoptosis was relatively frequent and in late pregnancy there was evidence of cell deletion by lysis. During pregnancy there was a slight increase in sinusoidal macrophages as a proportion of the total cell population but there did not appear to be significant changes in macrophage enzymic activity. It is suggested that the acid phosphatase activity present in macrophages makes a minor contribution to total liver activity, most of which is present in parenchymal cells. Acid phosphatase activity associated with single cell deletion appears to be quantitatively negligible. There was a direct relationship between total hepatic acid phosphatase activity and the numbers of peribiliary dense bodies, which were most numerous at the 15th day and at the end of gestation. It is suggested that these residual bodies contain products of detoxification processes and also cell structural elements resulting from enhanced liver metabolism and intracellular turnover during pregnancy.

The oral administration of an LD50 dose (25 mg/kg) of O,S,S-trimethyl phosphorodithioate to rats induced immediate, cholinergic symptoms. A delayed respiratory crisis followed, 3-4 days later, involving a pronounced increase in lung weight and extensive injury to the alveolar epithelium. This compound also induced the immediate liberation of secretory granules from the Clara cells. Minor changes in the surface appearance of these cells were also observed but no signs of injury were found in any cells of the bronchiolar epithelium. The complement of secretory granules was monitored, by the morphometric analysis of ultrathin sections. Clara cells from control animals were found to contain 9.55 +/- 1.16 (SEM) granules per cell profile. The Clara cells from dosed animals were largely devoid of granules until the 3rd day after administration. Many of these agranular cells were arranged in clusters and often showed signs of mitotic division. In surviving animals the subsequent replacement of granules resulted in a large increase in the numbers present and many exhibited abnormal morphology. Over twice the normal complement of granules, 22.45 +/- 0.42 (SEM) per cell profile, was found 6 days after dosing. The complement of granules subsequently returned to normal levels and the clusters of Clara cells were resolved within 14 days of dosing.

The subcellular distribution of calcium has been investigated in samples, from the intestinal mucosa of 10-day rats, prepared for X-ray microanalysis by various techniques designed to minimize the loss of this element. Calcium retention and its threshold of detection was most satisfactory in freeze-dried frozen thin sections. In resin-embedded samples the best retention of calcium was found in specimens fixed in absolute ethanol, embedded without osmication, and sectioned onto glycerol. The results of this investigation indicate the presence of calcium in the supranuclear vacuole of enterocytes in the distal intestine of the neonatal rat. This calcium is probably taken up during the endocytosis of material from the intestinal lumen. The same mechanism may also be important in the uptake of other metals by suckling animals.

Male Syrian hamsters were treated with hexachlorobenzene (HCB) in their diet at levels of 100 ppm for 28 weeks, 200 ppm for 18 and 28 weeks, and 500 ppm for 6 weeks. All treatments caused at least a 2.5- to 3-fold increase in thyroid size, mainly by enlargement of some follicles. Serum thyroxine (T4) levels were unchanged, whereas levels of triiodothyronine (T3) eventually became depressed by greater than 60%. Uptake of 131I into thyroids was induced approximately 3-fold when estimated after feeding HCB (500 ppm) for 3 or 6 weeks. Hamsters also lost weight by depletion of adipose tissue, leading to 50% mortality in longer experiments. Results were distinct from the effects of the known antithyroid agent 3-aminotriazole or amitrole (200 ppm for 28 weeks), which did not affect survival and although causing thyroid enlargement depressed serum T4 and significantly elevated T3. The effects of HCB in hamsters were also different from those in rats (500 ppm HCB for 6 weeks) in which there was only a small increase in thyroid size (1.3-fold), serum levels of T3 were only slightly depressed but T4 levels were reduced by 74%. These studies are discussed with reference to the effects of other polyhalogenated aromatic chemicals on the thyroid, serum thyroid hormone levels and lethality.

The i.v. administration of suspensions of beryllium phosphate (5-50 mumol/kg) to rats resulted in the vacuolation of hepatic Kupffer cells within 3 h. After 6 h necrotic Kupffer cells were common throughout the sinusoids of the liver but no changes were detected in the hepatic parenchymal cells during this period. A significant reduction in the numbers of intrasinusoidal cells was observed 14 h after treatment but this population had reverted to normal within 24 h. The administration of colloidal carbon to treated animals at this time did, however, demonstrate a reduction in the complement of functional endocytic cells. These results demonstrate a selective destruction of endocytic cells in the liver by this particulate toxin and the limited response by the organ to this injury. These observations are the most probable explanation for the reticuloendothelial blockade known to be caused in vivo by beryllium phosphate.

The fate of i.v. administered particles of beryllium phosphate (50 mumol/kg) has been investigated by electron microscopy in the liver of the rat. These particles were endocytosed by the Kupffer cells within 15 min of administration but they were never found in the endothelial or parenchymal cells of the liver. Endocytosed particles were rapidly degraded within vesicles which were identified as secondary lysosomes. Pronounced swelling of these lysosomes subsequently developed, within 60 min of administration, resulting in distension of the entire cell. This vacuolation was entirely restricted to the Kupffer cells, particularly those in the pericanalicular region of the hepatic lobules. Lysosomal swelling, or even rupture, probably resulted in the subsequent death of these cells by the intra-cytoplasmic release of beryllium.

In rats subcutaneous injection of colchicine resulted in sub-cellular necrosis within the rapidly proliferating cells in the duodenal mucosa. Similar lesions resulted from the administration of cytosine arabinoside hydrochloride and the effects of both drugs were ameliorated by treatment with cycloheximide. It is suggested that these lesions are a form of shrinkage necrosis involved in the deletion of growing stem-cells which, in the absence of mitotic division, results in mucosal atrophy.

The pathological effect of parainfluenza type I (Sendai virus) is known to be a bronchopneumonia, which becomes a chronic pneumonia in the immunodeficient athymic (nude) mouse. The severity of this established chronic pneumonia can be dramatically altered by providing the nude mouse with humoral monoclonal antibodies which are neutralizing, and are directed against the fusion protein, of the virus. The alveolitis, which is a significant part of the pathology, is suppressed due to a reduction (greater than 90%) in the number of virus-infected alveolar macrophages present in the alveoli. This clearly identifies the infected alveolar macrophage as the primary effector cell in the pathogenesis of alveolitis caused by parainfluenza virus type I. The implications of using virus-neutralizing monoclonal antibodies, which have little immunomodulatory toxicity, in the treatment of viral pneumonias are discussed.

Static exposure of rats to methyl isocyanate, for one hour, led to respiratory impairment during and after exposure. The resultant changes in breathing pattern suggest that this compound is both a sensory and a pulmonary irritant. Low concentrations resulted in extensive necrosis and loss of epithelial cells in the proximal airways but widespread alveolar damage was found only after exposure to high, lethal concentrations. At high concentrations methyl isocyanate probably caused peracute death through reflex inhibition of breathing. Animals surviving the exposure exhibited signs of airway narrowing and development of (haemorrhagic) pulmonary oedema. The epithelial lesions were repaired rapidly, but residual peribronchial fibrosis and signs of renewed injury/inflammation were apparent.

The localization of acid phosphatase activity was differentiated from that of alkaline phosphatase in the foregut of the newborn lamb by light and electron microscopy. The examination of samples from fed and unfed lambs indicated the presence of alkaline phosphatase activity in endocytic vesicles originating from the brush-border. These vesicles, associated with protein absorption, were particularly numerous in fed lambs and occurred throughout the cytoplasm of the enterocytes. Acid phosphatase activity was absent from vesicles in the apical cytoplasm but it was localized in most sub-nuclear vesicles, also in the Golgi apparatus and the lysosomes of macrophages. The sub-nuclear vesicles were often observed in close proximity to the lateral and basement membranes of the enterocytes, also in continuity with the intercellular space. It is suggested that these results indicate the mechanism for transmission of brush-border and lysosomal enzymes, along with the immunoglobulins, into the lymph of the newborn lamb.

The static exposure of rats to 0.25 mg/l methyl isocyanate for 1 h resulted in damage to the epithelium of the proximal bronchioles and upper airways. Bronchiolar cells exhibited both nuclear and cytoplasmic damage; many epithelial cells, particularly in the bronchi and trachea, were killed and/or dislodged from the basement membrane. A "raft" of cell debris and fibrin lined most of the airways during the 1st week after exposure but repair to the underlying epithelium was well advanced within 2-3 days. The majority of airways were lined by a normal epithelium within 3 weeks of exposure, but isolated foci of hyperplasia and occluded airways probably accounted for continued respiratory impairment.

Hexachlorobenzene (HCB) was fed to male and female F344 rats as 0.02% of the diet for 15 weeks. Females developed a massive porphyria, due to depression of uroporphyrinogen decarboxylase activity, whereas males did not. Although hepatic non-haem iron levels in control females were 3-5 times greater than males (iron is implicated in the pathogenesis of this condition) preloading the latter with iron did not increase their susceptibility. After 90 weeks of HCB treatment 100% of surviving females had multiple liver tumours which were strongly gamma-glutamyl transpeptidase (GGT) positive and histologically classified as neoplastic nodules or hepatocellular carcinomas. In contrast, only 16% of males developed tumours which were smaller and fewer in number per liver than those in females. Accumulation of porphyrins was still significantly less in males than females although no uroporphyrinogen decarboxylase activity was detected in treated livers of either sex. No differences in porphyrin levels or enzyme activity were found between tumours and surrounding tissue showing that tumours did not revert to a non-porphyric state. The sex difference in tumour response could not be explained by differences in hepatic HCB concentrations. Non-haem iron concentrations of livers fell after HCB treatment for 90 weeks in both sexes and were even lower in tumours. These studies demonstrate that not only are female rats far more sensitive than males to the porphyrinogenic effects of HCB but also to the hepatocarcinogenic actions, suggesting a link between these two manifestations of toxicity that may also apply to other polyhalogenated aromatics.