The role of cytosolic Ca2+ in cell injury, necrosis and apoptosis.

Increases in cytosolic Ca2+ are believed to be a pivotal signal in the regulation of cell injury, cell death, cell proliferation, cellular differentiation and cellular aging. Changes in the concentration of cytosolic Ca2+ are involved in both acute and chronic cell injury, as well as in accidental or programmed cell death. Signalling in all of these phenomena is dependent on mediated activities of a number of intracellular factors, including phospholipases, proteases and endonucleases. The coordinate regulation of these factors, as well as of oncogene activation, seems to play a role both in the processes of cell injury and cell death, and in the recovery from injury in sublethally injured cells.

Influence of glutaraldehyde and-or osmium tetroxide on cell volume, ion content, mechanical stability, and membrane permeability of Ehrlich ascites tumor cells.

Effects of fixation with glutaraldehyde (GA), glutaraldehyde-osmium tetroxide (GA-OsO(4)), and osmium tetroxide (OsO(4)) on ion and ATP content, cell volume, vital dye staining, and stability to mechanical and thermal stress were studied in Ehrlich ascites tumor cells (EATC). Among variables investigated were fixation time, fixative concentration, temperature, osmolality of the fixative agent and buffer, total osmolality of the fixative solution, osmolality of the postfixation buffer, and time of postfixation treatment in buffer (Sutherland, R. M., et al. 1967. J. Cell Physiol.69:185.). Rapid loss of potassium, exchangeable magnesium, and ATP, and increase of vital dye uptake and electrical conductivity occurred with all fixatives studied. These changes were virtually immediate with GA-OsO(4) or OsO(4) but slower with GA (in the latter case they were dependent on fixative temperature and concentration) (Foot, N. C. 1950. In McClung's Handbook of Microscopical Technique. 3rd edition. 564.). Total fixative osmolality had a marked effect on cell volume with OsO(4) but little or no effect with GA or GA-OsO(4). Osmolality of the buffer had a marked effect on cell volume with OsO(4), whereas with GA or GA-OsO(4) it was only significant at very hypotonic buffer osmolalities. Concentration of GA had no effect on cell volume. Osmolality of the postfixation buffer had little effect on cell volume, and duration of fixation or postfixation treatment had no effect with all fixatives. Freezing and thawing or centrifugal stress (up to 100,000 g) had little or no effect on cell volume after all fixatives studied. Mechanical stress obtained by sonication showed that OsO(4) alone produced poor stabilization and that GA fixation alone produced the greatest stabilization. The results indicate that rapid membrane permeability changes of EATC follow fixative action. The results are consistent with known greater stabilizing effects of GA on model protein systems since cells were also rendered relatively stable to osmotic stress during fixation, an effect not noted with OsO(4). After fixation with GA and/or OsO(4) cells were stable to osmotic, thermal, or mechanical stress; this is inconsistent with several earlier reports that GA-fixed cells retain their osmotic properties.

Extracellular acidosis protects Ehrlich ascites tumor cells and rat renal cortex against anoxic injury.

The present study indicates that extracellular acidosis protects Ehrlich ascites tumor cells and rat kidney cortex cells against injury from anoxia. Parameters measured included cell potassium, adenosine 5'-triphosphate, and uptake of vital dyes. Cells survived longer at a pH of 5.6 to 6.5 than at a pH of 7.4; pH 7.9 was most detrimental. These findings indicate that production of protons by anoxic cells may be a protective feedback mechanism.

Membrane alterations in hemolysis: Internalization of plasmalemma induced by primaquine.

Incubation of normal human erythrocytes with primaquine, a derivative of 8-aminoquinoline, results in internalization of the cell membrane and the formation of intracellular vacuoles. These changes are similar to those observed in other types of cells in pinocytosis. The reduction in surface cell membrane which accompanies internalization of plasmalemma may be generally significant in the destruction of red cells.

Microsomal lipid peroxidation: morphological characterization.

Lipid peroxidation of liver and kidney microsomes induces a highly characteristic sequence of morphological changes typified by detachment of ribosomes and formation of large aggregates of vesicles bound together by dense amorphous material and myelin figure-like debris. The trilaminar structure of the membrane is, however, retained even after complete peroxidation, though its spacing may be increased. The aggregates resemble lysosomal lipofuscin pigment as well as the membranous aggregates of endoplasmic reticulum seen in the liver after carbon tetrachloride poisoning.

Endoplasmic reticulum in rat renal interstitial cells: molecular rearrangement after water deprivation.

Cylindrical bodies in renal interstitial cells of dehydrated rats are confluent with membranes of endoplasmic reticulum. The cylinder walls, composed of helically arranged pentagonal tubules, may represent a molecular rearrangement of the membrane structure. The cylinders may represent a morphologic expression of altered ergastoplasmic function possibly related to the production of concentrated urine.

Interindividual variation in binding of benzo[a]pyrene to DNA in cultured human bronchi.

The binding of benzo[a]pyrene to DNA in cultured human bronchus was measured in specimens from 37 patients. The binding values ranged from 2 to 151 picomoles of benzo[a]pyrene per milligram of DNA with an overall mean +/- standard error of 34.2 +/- 5.2. This 75-fold interindividual variation in the binding of benzo[a]pyrene to DNA is similar in magnitude to that found in pharmacogenetic studies of drug metabolism. Aryl hydrocarbon hydroxylase is also inducible by benz[a]anthracene in the bronchial mucosa.

Transformation of human bronchial epithelial cells transfected by Harvey ras oncogene.

Transfection of normal human bronchial epithelial (NHBE) cells with a plasmid carrying the ras oncogene of Harvey murine sarcoma virus (v-Ha ras) changed the growth requirements, terminal differentiation, and tumorigenicity of the recipient cells. One of the cell lines isolated after transfection (TBE-1) was studied extensively and shown to contain v-Ha ras DNA. Total cellular RNA from TBE-1 cells hybridized to v-Ha ras structural gene fragment probes five to eight times more than RNA from parental NHBE cells. The TBE-1 cells expressed phosphorylated v-Ha ras polypeptide p21, showed a reduced requirement for growth-factor supplements, and became aneuploid as an early cellular response to v-Ha ras expression. As the transfectants acquire an indefinite life-span and anchorage independence they became transplantable tumor cells and showed many phenotypic changes suggesting a pleiotropic mechanism for the role of Ha ras in human carcinogenesis.

Induction of ouabain-resistant mutation and sister chromatid exchanges in Chinese hamster cells with chemical carcinogens mediated by human pulmonary macrophages.

Pulmonary macrophages (PAM) metabolically activated benzo[a]pyrene [B(a)P] and its proximate carcinogenic metabolite, (+/-)trans 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (7,8-diol), to ultimate mutagens that were detected in cocultivated Chinese hamster V79 cells. Increases in the frequency of ouabainresistant (O(r)) mutations and sister chromatid exchanges were found in V79 cells only when they were cocultivated with both PAM and the chemical procarcinogens. 7,8-Diol caused higher frequencies of both O(r) mutations and sister chromatid exchanges than did the parent compound, B(a)P. When metabolically activated by PAM the mean O(r) mutation frequency caused by B(a)P was 9 O(r) mutants/10(6) surviving V79 cells per 10(6) PAM and a 10-fold interindividual variation (range, 2-21) was found. The mean O(r) mutation frequency caused by 7,8-diol was 64 and a ninefold interindividual variation (range, 14-120) was found. In the absence of PAM, the O(r) mutation frequency in V79 cells was one or less O(r) mutant per 10(6) survivors. 7,8-Benzoflavone, an inhibitor of mixed function oxidases, reduced the frequencies of O(r) mutations and of sister chromatid exchanges in V79 cells caused by 7,8-diol and B(a)P. As expected 7,8-benzoflavone did not influence the frequency of O(r) mutations caused by one of the ultimate mutagens derived from B(a)P and 7,8-diol, (+/-)7beta, 8alpha-dihydroxy-9alpha, 10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. These data are consistant with the hypothesis that PAM may play a role in the activation of environmental chemical procarcinogens.

Colon epithelium. I. Light microscopic, histochemical, and ultrastructural features of normal colon epithelium of male Fischer 344 rats.

Although the colon of the inbred F344 rat is not distinctly demarcated into ascending, transverse, and descending segments as in the human colon, it can roughly be divided into ascending and descending portions that show distinct light microscopic, histochemical, and ultrastructural features. The ascending colon is characterized by a "herringbone" pattern of mucosal folds and test-tube-shaped uniform crypts that contain mucous cells (MC) with abundant mucin (acidic mucopolysaccharide--mostly sialomucin) in the lower one-third of the crypt, whereas the upper one-third contains two putative cell populations: 1) MC containing large globules of neutral mucopolysaccharide or sulfomucin and 2) columnar cells (CC), the full capabilities of which are unknown. The descending colon has longitudinal folds and contains sparse MC with small mucous granules at the lower one-third of the crypt, whereas the upper one-third contains numerous goblet cells. Neutral mucopolysaccharide is sparse and the acidic mucin is exclusively sulfated. Histochemically, the descending segment of the rat colon resembles the human descending colon in that the predominant type of mucus is sulfomucin. Ultrastructurally, the cell types observed in both the ascending colon and the descending colon are: a) MC, b) CC, c) endocrine cells, and d) undifferentiated cells.

Colon epithelium. III. In vitro studies of colon carcinogenesis in Fischer 344 rats. N-methyl-N'-nitro-N-nitrosoguanidine-induced changes in colon epithelium in explant culture.

Colon explants from the inbred F344 rat descending colon pretreated in vivo with azoxymethane and maintained in explant culture were exposed to the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). One week after the MNNG treatment, the colon crypts showed marked crowding, hypercellularity, and stratification of cells. Nine weeks after the treatment, the explants showed epithelial papillary projections on the surface epithelium and within the crypts, in addition to hypercellularity and stratification. The control untreated explants maintained a single layer of epithelium during the entire culture period. Ultrastructurally, the treated cells showed an unusual concentration of free polysomes and thin and thick filaments, multiple and bizarre nucleoli, nuclear indentations and pseudoinclusions, and intracellular lumina. Sulfomucin was the predominant component in the control untreated explants as well as in the normal descending colons of rats and humans. One week after treatment the crypts of the carcinogen-treated explants showed an increase in sialomucin, and by 9 weeks after treatment, they showed mostly sialomucin. These features, compared and correlated with those of the parallel in vivo animal model as well as with human material, lend additional support to de novo histogenesis of colon carcinoma.

Comparative study of the morphologic, histochemical, and proliferative changes induced in the large intestine of ICR/Ha and C57BL/Ha mice by 1,2-dimethylhydrazine.

The mouse model of 1,2-dimethylhydrazine (DMH)-induced colorectal carcinogenesis was studied to determine the susceptibility of different anatomic segments of the large intestine in ICR/Ha (susceptible) and C57BL/Ha (resistant) mice. In ICR/Ha mice numerous exophytic macroscopic neoplasms were found in the distal colon and rectum after 15 weekly injections of DMH (20 mg/kg). The proximal colon was free of any microscopic or macroscopic neoplasms. In contrast, C57BL/Ha mice given the same treatment showed no macroscopic neoplasms. However, foci of dysplastic crypts were observed throughout the large intestine of C57BL/Ha mice with highest incidence in the distal colon and rectum. In some areas dysplastic crypts were clearly invading the muscularis mucosae and were, therefore, microscopic carcinomas (microcarcinomas). Thus C57BL/Ha mice were not totally resistant to the neoplastic stimulus of DMH, and the susceptibility of the large intestine is site-specific in both mouse strains.

Induction and properties of aryl hydrocarbon hydroxylase in bovine pancreatic ducts.

Inducibility and characteristics of aryl hydrocarbon hydroxylase (AHH) in cultured bovine pancreatic ducts were studied by the fluorometric method. AHH was present and inducible in all the pancreatic ducts studied when they were exposed to 20 microgram benz[a]anthracene (BA)/ml medium. AHH activity in the control tissue ranged from 1.0 to 3.0 U/mg DNA, whereas the activity in the BA-treated tissue was 4.2--28.5 U/mg DNA, which resulted in the induction of 5- to 18-fold activity. At 12 hours of BA exposure, AHH activity in the treated tissue was 12-fold that in the control tissue and continued to increase to 15-, 19-, and 31-fold that in the control tissue at 24, 48, and 72 hours, respectively. The BA-induced AHH activity had a broad pH optimum between 7.1 and 7.7, and the maximum activity was found at pH 7.4. The AHH activity was linear with respect to the incubation time up to 30 minutes. The effect of the benzo[a]pyrene concentration on AHH activity was studied on the BA-induced enzyme. The apparent Michaelis constant for the substrate was 0.5 microM, and the maximum velocity was 8.6 U/mg DNA. BA-induced AHH activity was inhibited 65% by 100 microM 7,8-benzoflavone, whereas the control enzyme activity was stimulated 100% by the same concentration of 7,8-benzoflavone.

Aryl hydrocarbon hydroxylase in human bronchial epithelium and blood monocyte.

The inducibility of aryl hydrocarbon hydroxylase (AHH) in human bronchial epithelium and blood monocyte was studied in 11 immediate autopsy patients without lung cancer. When the bronchus was exposed to 10 microgram of benz[a]anthracene (BA)/ml medium in explant culture, the levels of AHH induction in the bronchus were 3- to 29-fold above control levels. The specific enzyme activity ranged from not detectable (i.e., < 0.14) to 2.9 nmol/hour/mg DNA in untreated tissue and from 1.2 to 30 nmol/hour/mg DNA in BA-treated bronchus. The optimum pH for the bronchus AHH was 7.7. Control AHH and BA-induced AHH in bronchus were both inhibited by 100 microM 7,8-benzoflavone in vitro. Induction of AHH in monocytes ranged from 1.5- to 30-fold above that of controls when the cells were exposed to 2 microgram of BA/ml medium in culture. The specific enzyme activity ranged from 1.6 to 19 pmol/hour/10(6) cells in untreated monocytes and from 5.8 to 53 pmol/hour/10(6) cells in BA-treated monocytes. The extent of AHH induction in monocytes depended on BA concentration (from 0.1 to 10.0 microgram) in a dose-related manner. AHH activity increased linearly with the number of monocytes (from 0.5 to 2x10(6)) in the assay system. 7,8-Benzoflavone inhibited the BA-induced but not the basal levels of monocyte AHH activity. The data are consistent with a correlation between the inducibility of AHH in human bronchus and blood monocyte from the same individual.

The respiratory epithelium. II. Hamster trachea, bronchus, and bronchioles.

The normal female hamster respiratory epithelium at five airway levels was characterized with the use of coordinated morphologic and histochemical techniques. Five morphologic cell types were recognized in the trachea, stem bronchi, and primary bronchl: basal cells and neurosecretory cells that were basally located and did not reach the lumen and mucous cells [mucous goblet cells and small mucous granule cells (SMGC)], indifferent cells showing mucous-ciliary differentiation, and ciliated cells that reached the lumen. Two epithelial cell types were observed in the bronchioles, ciliated cells and nonciliated Clara cells, both of which reached the lumen. Mucous cells presented as either SMGC with a few small periodic acid-Schiff-positive granules (diastase-resistant neutral mucosubstances) or as goblet cells, filled with the same material. Mucous cells were columnar, and the cytoplasm was electron-dense and contained a well-developed endoplasmic reticulum and Golgi complex. The microvilli of the mucous cells were coated more thickly with colloidal iron than either the cilia or microvilli of ciliated cells. Approximately one-half the cells in the trachea, bronchi, and bronchioles were ciliated. Ciliated cells containing intracellular ciliated cysts with normal cilia projecting into a closed space or ciliated cells bearing compound cilia were observed infrequently. Neurosecretory cells were rarely observed. These cells contained characteristic dense-core granules.

The respiratory epithelium. VI. Histogenesis of lung tumors induced by benzo[a]pyrene-ferric oxide in the hamster.

Lung tumors were induced in female Syrian golden hamsters by intratracheal instillation of benzo[a]pyrene-Fe2O3. The tumors were characterized with the use of coordinated morphologic and histochemical techniques including electron microscopy. The lung carcinomas were classified according to their presumed cell of origin. Most were derived from mucous cells and/or basal cells, and they were classified as either epidermoid carcinomas or as combined epidermoid and adenocarcinomas. The tumors in the second group (57% of the total number of carcinomas) presented a wide spectrum of epidermoid and adeno components. The epidermoid component was characterized in well-differentiated tumors by the presence of intercellular bridges and/or keratinization. Well-developed desmosomes and numerous bundles of tonofilaments were observed ultrastructurally. In diagnosing adenocarcinoma, one no longer needs to depend on the presence of tubules or gross glandular structures as criteria for diagnosis. The presence of intracellular and/or extracellular alveoli, well-developed Golgi complex, and endoplasmic reticulum and/or evidence of mucous secretion provide more definitive criteria. A tumor composed of neurosecretory cells that morphologically resembled a bronchial carcinoid of man was observed. Nests of uniform, small, polygonal cells with round-to-oval nuclei were seen at the light microscopic level. Dense-core secretory granules 1,100-2,200 A were present in the cytoplasm of the tumor cells. Several fibrosarcomas were observed. The tumors showed a very cellular structure, composed of either densely packed ovoid or spindle-shaped cells. Ultrastructurally, the cells resembled fibroblasts. The results obtained in this study give strong support for a histogenetic classification, i.e., a classification based on the cell of origin.

Studies on carcinogenesis of human prostate. III. Long-term explant culture of normal prostate and benign prostatic hyperplasia: transmission and scanning electron microscopy.

Ultrastructural changes in normal human prostate and benign prostatic hyperplasia (BPH) during long-term explant culture were compared. Explants of normal prostate obtained at immediate autopsy of young adults of BPH obtained at the time of surgery were maintained as long as 24 weeks in vitro. Ultrastructural changes occurring in epithelial cells during culture were monitored by transmission and scanning electron microscopy. Essentially identical results were found for normal prostate and BPH. During the first week of culture, secretory epithelial cells degenerated and sloughed into the acinar lumen, resulting in an accumulation of necrotic debris. During this period, however, epithelial cells with ultrastructural characteristics of basal cells remained viable, repopulated glandular structures, migrated from glands and ducts, and epithelialized adjacent cut surfaces, eventually covering the explant. On explant surfaces, these basal cells initially were squamous-like, but they later became typically cuboidal, polygonal, or sometimes columnar and formed an epithelium, two cells or more thick. Epithelium with similar features lined acini within explants. Epithelial cells at the surface or within explants were distinguished by the presence of microvilli, junctional complexes, multiple Golgi complexes, well-developed rough endoplasmic reticulum, polyribosomes, nuclei with prominent nucleoli, orthodox mitochondria, scattered tonofilaments, and a basal lamina. Some epithelial cells extended from the lumen to the basal lamina; others were oriented along the basal lamina and did not extend to the lumen. By 1--2 weeks in vitro, these epithelial cells began synthesis of mucus-like material. At later intervals of culture, microvilli were shortened and mucosubstances were reduced. During culture, the stroma became progressively hypocellular and necrotic. In summary, explant-cultured epithelial cells of normal human prostate or BPH were similar ultrastructurally and were found to originate from basal cells, which alone survive culture conditions.

N-Methyl-N-nitrosourea and saccharin: effects on epithelium of normal human urinary bladder in vitro.

The effects of N-methyl-N-nitrosourea (MNU) and saccharin on the histology of normal human bladder "urothelium" (i.e., the epithelium of the urinary bladder) were studied in long-term explant cultures. In MNU-treated cultures, a dose response was observed. Single doses of 1-100 micrograms MNU/ml induced atypical focal hyperplasia, which reverted to a single or a double cell layer as seen in controls. Exophytic, papillary-like hyperplastic structures were noted after a single dose of 10 or 100 micrograms MNU/ml. In contrast to single doses, multiple doses (given every 2 wk) of MNU at 5 or 10 micrograms/ml resulted in striking focal proliferation of dysplastic spindle cells in as little as 6 weeks (three doses of MNU). At 0.5% saccharin, urothelium on explant surfaces resembled that of controls, except in one instance in which mild focal hyperplasia persisted. In the presence of saccharin, hyperplasia induced by a single dose of MNU persisted. Following three multiple doses of MNU in the presence of saccharin, spindle cell hyperplasia was induced similar to that seen with multiple doses of MNU alone, although nuclei appeared more pleomorphic and hyperchromatic in the presence of saccharin.

Adenocarcinoma of the kidney. IV. Electron microscopic study of the development of renal adenocarcinomas induced in rats by N-(4'-fluoro-4-biphenylyl)acetamide.

Ultrastructural features of various stages in the histogenesis of renal adenocarcinoma induced in F344 rats by the carcinogen N-(4'-fluoro-4-biphenylyl)acetamide (FBPA) are described. FBPA was added to the diet up to 48 weeks; animals were killed at intervals from 4 to 52 weeks. Characterized were foci of cellular alterations followed by foci of proliferation with dysplasia leading to solid and cystic lesions, carcinoma in situ, and finally, carcinoma. Other lesions included foam cell nodules. Alterations also were noted in distal tubules. By electron microscopy, subtle changes in some proximal tubule cells of the pars recta were noted by 4 weeks (foci of cellular alteration). By 12 weeks there was evidence of dedifferentiation and reduction of cell polarity, thickening of basal lamina, alterations in endoplasmic reticulum and mitochondria, and increases in residual bodies. Nuclei of these cells possessed large nucleoli. From 24 to 36 weeks foci of proliferation were seen and consisted of tumor cells arranged in solid and cystic configurations. In solid lesions (less than 3 mm), in addition to the above alterations, cells showed a microvillus brush border in unusual locations, small and irregular mitochondria, multiple Golgi complexes and basal lamina, and loss of cell polarity. From 36 to 52 weeks carcinoma in situ consisted of cells similar to tumor cells described above. In addition, intracellular canaliculi were present. Areas of tumor cell degeneration and necrosis also were seen. Cells of larger carcinomas (greater than 3 mm), also observed at this interval, were identical to those of carcinoma in situ. Calcification of necrotic debris was noted.

Adenocarcinoma of the kidney. I. Ultrastructure of renal adenocarcinomas induced in rats by N-(4'-fluoro-4-biphenylyl)acetamide.

The ultrastructure of tumors induced in kidneys of F344 rats by the carcinogen N-(4'-fluoro-4-biphenylyl)acetamide appeared similar in many aspects to that described in humans and other animal models. The neoplastic cells exhibited microvilli resembling brush border and other adaptations of the cell membrane. Tumor lobules were surrounded by basement membrane-like material, which also extended into the tumor mass between individual cells. The ultrastructure appeared to reflect a proximal tubular origin of these lesions.