Midzonal Regions Research Articles

Shifting necrosis: butylated hydroxytoluene (BHT) and phenobarbital move cocaine-induced hepatic necrosis across the lobule

Cocaine (60 mg/kg i.p.) caused centrilobular necrosis in the livers of 55 % of DBA/2Ha mice. Pretreatment with phenobarbital (PB, 3 × 80 mg/kg i.p.) increased the incidence of necrosis to 70 % and shifted this damage to the midzonal region. Pretreatment with butylated hydroxytoluene (BHT, 0.1 % in diet) increased the severity of the centrilobular damage and increased the incidence to 90%. Combined treatment with both PB and BHT shifted the site of necrosis to the periportal region of the liver, and induced necrosis in all animals. Microsomal malondialdehyde (MDA) did not reflect the extent of the damage and/or correlate with the site of damage. These results argue against a causal role for lipid peroxidation in the mechanism of cocaine hepatotoxicity, and demonstrate that prior exposure to enzyme-inducing agents can increase sensitivity and dramatically alter the site of cell damage.

Pathophysiology of sea nettle (Chrysaora quinquecirrha), envenomation in a rat model and the effects of hyperbaric oxygen and verapamil treatment

The effect of lethal sea nettle envenomation on the morphology and blood flow in various rat organs was characterized and the influence of two antidotes (hyperbaric oxygen and verapamil) was compared. Either antidote slightly prolonged survival, but the protective effects were not statistically significant. The venom caused no histologic alterations in brain, heart, or lungs but induced hepatic and renal necrosis. Hepatocytes in mid-zonal regions and renal tubular epithelium were the cell types predominantly affected. Hyperbaric oxygen and verapamil did not decrease the hepatic injury. The venom did not influence central hemodynamics until preterminally and it diminished blood flow to brain, but not to liver or kidney. Hyperbaric oxygenation protected against venom-induced decreases in blood flow to the brain. These results add toxic hepatic and renal necrosis and cerebral ischemia to the pathophysiology of envenomation in this model.

Immunofluorescent analysis of γ-glutamyl transpeptidase and glutathione-S-transferase-P during the initial phase of experimental hepatocarcinogenesis

We used double-label immunofluorescence to evaluate the expression of glutathione-S-transferase-P (GST-P) and gamma-glutamyl transferase (gamma GT) in individual liver cells in rats placed on a modified Solt-Farber hepatocarcinogenesis protocol. Four days after treatment with diethylnitrosamine (DEN), single GST-P+ cells were found in the centrilobular and midzonal regions. Most of these were gamma GT-, but the percentage of gamma GT+ cells increased with DEN dose to a high of 32%. At later times (7-8 days) doublets and small clusters of GST-P+ cells predominated; all cells in each pair or cluster displayed the same phenotype (GST-P+ or GST-P+/gamma GT+). Following administration of acetylaminofluorene and partial hepatectomy, lesions were larger and a greater percentage were GST-P+/gamma GT+. However, within these lesions individual cells displayed different phenotypes. These results indicate that the expression of GST-P and gamma GT is discordant in individual cells and small clusters soon after treatment, and suggest that different molecular events may be involved in their expression.

Zonal heterogeneity of peroxisome proliferation and morphology in rat liver after gemfibrozil treatment.

The effect of gemfibrozil on the fine structure of peroxisomes across the rat liver lobule was investigated by light and electron microscopy using the alkaline diaminobenzidine (DAB) medium for the visualization of catalase peroxidatic activity. The oral administration of gemfibrozil for 2 weeks induces a striking heterogeneity in the lobular distribution of peroxisomes. The size and shape of peroxisomes, variety of matrix modifications, catalase content, and position within the cell, are functions of the zonal localization of the hepatocytes. The largest and most numerous peroxisomes were found in the centrilobular region indicating that these cells are most sensitive to peroxisome proliferation. On the other hand, the greatest variety of peroxisome shapes and matrix alterations (tubules and plates) was seen more peripherally in the mid-zonal and periportal regions. The larger, round centrilobular peroxisomes stained less intensely than the elongated peroxisomes found more peripherally, indicating a discrepancy between peroxisome size and catalase content. A distinct population of small irregularly shaped peroxisomes, lacking matrix specializations and containing variable catalase content, was found in the mid-zonal region. Peroxisomes in the centrilobular region were located within areas of the cell containing SER and glycogen while those in the more peripheral region were relegated to areas of the cytoplasm separate from RER and SER. In addition to modifications of peroxisomes, gemfibrozil treatment resulted in a proliferation and formation of whorled configurations of SER. This was particularly evident in the mid-zonal region, where single peroxisomal profiles could be seen surrounded by whorls of SER membranes. The results suggest that rat liver hepatocytes of the centrilobular region are the most sensitive to peroxisome proliferation and those of the periportal area are most susceptible to peroxisome matrix alterations after gemfibrozil treatment.

Early midzonal cell death during low-flow hypoxia in the isolated, perfused rat liver: protection by allopurinol.

Trypan blue uptake and lactate dehydrogenase release were measured as indices of irreversible cell damage in isolated, perfused rat livers during low-flow hypoxia. In livers from fasted rats perfused in the anterograde direction, trypan blue uptake took place beginning at about 45 min of hypoxia. Cells which took up trypan blue first were located in narrow bands at the border between anoxic pericentral areas and normoxic periportal regions of the liver lobule. After longer periods of hypoxia, trypan blue uptake progressed towards the central vein until after 120 min virtually all cells in the pericentral regions were stained. Under these conditions, cells in periportal regions were spared. In perfusions in the retrograde direction, cell death began in midzonal regions and spread towards the portal vein. Release of lactate dehydrogenase into the effluent paralleled trypan blue uptake, beginning at about 40 min of low-flow hypoxia and peaking at 80 min. In contrast to livers from fasted rats, trypan blue was not taken up, and lactate dehydrogenase was not released in livers from fed rats exposed to low-flow hypoxia for as long as 120 min. To test the hypothesis that xanthine oxidase-mediated oxygen-free radical formation was involved in cell injury at the border between anoxic and normoxic regions (anoxic edge), allopurinol, an inhibitor of xanthine oxidase, was studied. Allopurinol (0.2 to 5 mM) delayed the release of lactate dehydrogenase during low-flow hypoxia in a dose-dependent fashion (e.g., 5 mM allopurinol delayed hypoxia-induced lactate dehydrogenase release by about 30 min).(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatotoxicity of the synthetic oestrogen hexoestrol in the female rat.

The synthetic oestrogen hexoestol is hepatotoxic to the female rat. Twenty-five animals were treated daily with hexoestrol at a dose of 60 mg/kg. Twenty-five more acted as controls. Five animals from each group were killed after 4, 12, 20, 30 and 40 days of treatment to permit serial evaluations of liver histopathology to complement serum biochemical investigations. There were no mortalities during the study and only modest external signs of toxicity. All the treated rats showed reductions in body weight and appetite and gains in liver weight. This latter effect was due to both cellular hypertrophy and hyperplasia. The principle finding of the liver histopathology was fatty change. This affected scattered individual cells, mainly in the midzonal region. All of the serum parameters of toxicity--which consisted of bilirubin, total protein, albumin, glycoprotein, alpha 2-macrofoetoprotein, and alkaline phosphatase--indicated liver impairment. Most of these also showed time-related changes consistent with an initial phase up to Day 20 of marked liver dysfunction superseded by a more adaptive phase thereafter. The hepatotoxicity described here seems sufficient to explain blood clotting defects observed elsewhere in oestrogen-treated rats.

Polychlorinated biphenyls, classified as either phenobarbital- or 3-methylcholanthrene-type inducers of cytochrome P-450, are both hepatic tumor promoters in diethylnitrosamine-initiated rats

The cytochrome P-450 isozymes, cytochrome P-450 MC 1 and MC 2, purified from rats treated with 3-methylcholanthrene (MC), were found by immunohistochemical staining to be strongly induced in the livers of rats treated with 3,3′, 4,4′-tetrachlorobiphenyl (TCBP), while the cytochrome P-450 isozymes, PB 1 and PB 2, purified from the livers of rats treated with phenobarbital (PB), were shown to be induced in the livers of rats treated with 2,2′, 4,4′, 5,5′-hexachlorobiphenyl (HCBP). The latter compound also strongly induced NADPH-cytochrome P-450-reductase. Following induction, all 5 enzymes were located preferentially in the centrilobular and midzonal region of the liver acinus. The influence of these polychlorinated biphenyls (PCBs) on diethylnitrosamine (DEN)-initiated hepatocarcinogenesis was investigated by analyzing the evolution of adenosine triphosphatase-deficient focal lesions. Whereas DEN alone produced very few islets, the administration of either PCB congener (150 μmol/kg, i.p., once weekly over a period of 8 weeks) subsequent to DEN treatment (50 ppm in the drinking water, 10 days) strongly enhanced the number of islets as well as the relative volume of liver occupied by islet tissue. These effects were evident, both 1 and 9 weeks, after cessation of PCB treatment. Unexpectedly the less persistent PCB congener, TCBP, showed a much more potent enhancing effect after the 9 weeks recovery period than did (HCBP).

Chronic Toxicity and Oncogenicity Study on Inhaled Vinylidene Chloride in Rats

Chronic Toxicity and Oncogenicity Study on Inhaled Vinylidene Chloride in Rats. QUAST, J. F., MCKENNA, M. J., RAMPY, L. W., AND NORRIS, J. M. (1986). Fundam. Appl. Toxicol. 6, 105–144. Male and female Sprague—Dawley rats (Spartan substrain) were exposed to vinylidene chloride (VDC) by inhalation for 18 months to assess chronic toxicity and oncogenic potential of the subject test material. Interim sacrifices were performed at 1, 6, and 12 months. Rats were exposed to VDC concentrations of 10 and 40 ppm for 6 hr/day, 5 days/week for the first 5 weeks of the study. Based upon the absence of observable treatment-related effects among rats sacrificed after 1 month of exposure, the exposure concentrations were increased to 25 and 75 ppm VDC. Exposures were continued at these concentrations through the 18th month of the study after which the surviving animals were held until 24 months and then sacrificed. Cytogenetic evaluations were performed on a separate group of animals, four rats/sex, exposed to 0, 25, or 75 ppm VDC for 6 months. There were no exposure-related changes in the following parameters: mortality, appearance and demeanor, body weight data, clinical chemistry determinations, hematologic evaluations, urinalysis, or cytogenetic evaluation of bone marrow preparations. A target organ effect, characterized by hepatocellular fatty change in the midzonal region of the hepatic lobule which was minimal in severity, was observed in both male and female rats of both the 25- and 75-ppm exposure groups as early as the 6-month interim sacrifice. The midzonal fatty change was also observed at the 12-month sacrifice but no indication of progression of this lesion in either severity or incidence was apparent. During the last 6 months of the study, after exposures had been discontinued, this effect was no longer discernible; therefore this alteration was readily reversible. The incidences of several tumors and/or tumor types were statistically increased or decreased in VDC-exposed rats when compared to their respective control groups; none of these differences were judged to be attributable to VDC exposure.

Chronic toxicity and oncogenicity study on inhaled vinylidene chloride in rats

Male and female Sprague-Dawley rats (Spartan substrain) were exposed to vinylidene chloride (VDC) by inhalation for 18 months to assess chronic toxicity and oncogenic potential of the subject test material. Interim sacrifices were performed at 1, 6, and 12 months. Rats were exposed to VDC concentrations of 10 and 40 ppm for 6 hr/day, 5 days/week for the first 5 weeks of the study. Based upon the absence of observable treatment-related effects among rats sacrificed after 1 month of exposure, the exposure concentrations were increased to 25 and 75 ppm VDC. Exposures were continued at these concentrations through the 18th month of the study after which the surviving animals were held until 24 months and then sacrificed. Cytogenetic evaluations were performed on a separate group of animals, four rats/sex, exposed to 0,25, or 75 ppm VDC for 6 months. There were no exposure-related changes in the following parameters: mortality, appearance and demeanor, body weight data, clinical chemistry determinations, hematologic evaluations, urinalysis, or cytogenetic evaluation of bone marrow preparations. A target organ effect, characterized by hepatocellular fatty change in the midzonal region of the hepatic lobule which was minimal in severity, was observed in both male and female rats of both the 25- and 75-ppm exposure groups as early as the 6-month interim sacrifice. The midzonal fatty change was also observed at the 12-month sacrifice but no indication of progression of this lesion in either severity or incidence was apparent. During the last 6 months of the study, after exposures had been discontinued, this effect was no longer discernible; therefore this alteration was readily reversible. The incidences of several tumors and/or tumor types were statistically increased or decreased in VDC-exposed rats when compared to their respective control groups; none of these differences were judged to be attributable to VDC exposure.

Immunofluorescent determination of the lobular distribution of a constitutive form of hepatic microsomal cytochrome P-450.

We have used an immunohistochemical approach to study the lobular distribution of constitutive liver microsomal cytochrome P-450. Cytochrome P-450 isolated (10.3 nmoles per mg protein) from hepatic microsomes from untreated, mature male Sprague-Dawley rats was used to produce antisera in rabbits. The IgG fraction produced single precipitin lines of identity with liver microsomes after double immunodiffusion, precipitated 80% of the total microsomal cytochrome P-450 and inhibited three cytochrome P-450-dependent enzyme activities. By these criteria, the IgG appeared to be specific for a constitutive form (or immunochemically related family) of liver microsomal cytochrome P-450. The pattern of fluorescence after indirect immunofluorescent labeling of liver sections depended on the route of tissue preparation and the concentration of primary antibody. In frozen sections, the labeling was uniform throughout the lobule, whereas in "antigen-depleted" paraffin-embedded sections it was heaviest in the centrilobular and midzonal regions. Increasing the concentration of primary antibody to 500 micrograms per ml inhibited the centrilobular labeling in frozen sections in a concentration-dependent manner. When specific isozymes of cytochrome P-450 were induced with phenobarbital or 3-methylcholanthrene, the constitutive cytochrome P-450 was localized predominantly in the periportal region. Decreases in cytochrome P-450 in rats treated with carbon tetrachloride or 1,2-dibromo-3-chloropropane were associated with antigen loss only in necrotic cells. Regional differences in the loss of antigen in paraffin sections and the inhibition of fluorescence in frozen sections establish that the lobular distribution of constitutive hepatic microsomal cytochrome P-450 is qualitatively heterogeneous and may be altered during hepatocellular responses to chemical treatment.

Ultrastructural changes in migrating leukocytes in the liver following lethal E. coli bacteremia in rats

Hepatocellular injury and dysfunction after Gram-negative sepsis have been well recognized in both humans and experimental animal models (1,2) with circulating septic components being attributed to producing the hepatocellular injury. We have previously shown, in a lethal E. coli bacteremic rat model, the progressive migration of leukocytes and foci of hepatic lesions along with release of liver enzymes (2). In those studies, hepatic lesions were randomly scattered throughout the liver but were prominent in midzonal regions where migrated and aggregated leukocytes were frequently seen, suggesting that there may be some correlation between the presence of leukocytes and damage to hepatic parenchymal cells (2). In the same model, infiltration of leukocytes into the myocardial interstitium was also noted (3), possibly contributing to the observed myocardial dysfunction.

Abnormalities in liver iron accumulation during N-2-fluorenylacetamide hepatocarcinogenesis that are dependent or independent of continued carcinogen action.

The characteristics of liver iron accumulation were studied during N-2-fluorenylacetamide (FAA)-induced hepatocarcinogenesis in rats. After injection of iron-dextran in control rats, hepatocytes accumulated stainable iron evenly throughout hepatic lobules. During the feeding of FAA, iron accumulation was reduced in the midzonal and centrilobular regions. After FAA removal, hepatocytes in these regions again accumulated high amounts of iron. Hepatocellular altered foci induced by FAA displayed rather uniform (> 94%) iron-exclusion during FAA feeding. After FAA removal, however, iron-exclusion was lost in a fraction of the foci, while others (40-64%) remained resistant to iron accumulation. A large majority of liver neoplasms (> 93%) displayed resistance to cellular iron accumulation both during FAA feeding and after removal of FAA. Thus, iron-exclusion by liver neoplasms is carcinogen-independent and irreversible, in contrast with that of normal hepatocytes which is completely carcinogen-dependent and reversible. Altered foci appear to represent two populations: one is characterized by reversible iron-exclusion whereas the other, like neoplasms, possesses permanent iron-exclusion.

Quantitative immunohistochemistry: a comparison of microdensitometric analysis of unlabeled antibody peroxidase-antiperoxidase staining and of microfluorometric analysis of indirect fluorescent antibody staining for nicotinamide adenosine dinucleotide phosphate (NADPH)-cytochrome c (P-450) reductase in rat liver.

The intralobular distribution of nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome c (P-450) reductase (NADPH:ferricytochrome oxidoreductase, EC 1.6.2.4) in rat liver has been investigated by means of two quantitative immunohistochemical techniques: microdensitometric quantitation of unlabeled antibody peroxidase-antiperoxidase staining and microfluorometric analysis of indirect fluorescent antibody staining. Utilizing sheep antiserum elicited against NADPH-cytochrome c (P-450) reductase that had been isolated and purified to apparent homogeneity from rat liver microsomes, the reductase was detected within hepatocytes throughout the liver. However, differences in the intensity of staining of hepatocytes within different regions of the liver lobule were readily apparent after completion of both immunohistochemical staining procedures. These visual findings were verified by microdensitometric and microfluorometric analyses of immunohistochemical staining, both of which revealed that approximately the same degree of staining for NADPH-cytochrome c (P-450) reductase was produced within the centrilobular and midzonal regions of the liver lobule, whereas periportal hepatocytes were stained with significantly less intensity. These results demonstrate that the application of either microdensitometry in conjunction with unlabeled antibody peroxidase-antiperoxidase staining or microfluorometry after indirect fluorescent antibody staining can be used to quantitatively determine the intratissue distributions of antigens.

Effects of phenobarbital, trans-stilbene oxide, and 3-methylcholanthrene on epoxide hydrolase within centrilobular, midzonal, and periportal regions of rat liver.

The effects of phenobarbital, trans-stilbene oxide, and 3-methylcholanthrene on epoxide hydrolase (EC 3.3.2.3) within centrilobular, midzonal, and periportal hepatocytes were investigated employing rabbit anti-serum produced against rat hepatic microsomal epoxide hydrolase in unlabeled antibody peroxidase-anti-peroxidase and indirect fluorescent antibody-staining techniques. In livers of control rats, midzonal and periportal hepatocytes bound the anti-epoxide hydrolase to similar extents while centrilobular hepatocytes bound approximately 25% more antibody. 3-Methylcholanthrene did not cause significant alterations in immunohistochemical staining for epoxide hydrolase within any region of the liver lobule, whereas phenobarbital and trans-stilbene oxide produced significant alterations in both the intensity and pattern of intralobular staining for the enzyme. After 4 days of phenobarbital pretreatment, anti-epoxide hydrolase binding to hepatocytes was slightly, but significantly, elevated, especially within midzonal regions. After 7 days of phenobarbital pretreatment, anti-epoxide hydrolase binding was increased by approximately 65% within midzonal regions and by approximately 41 and 24%, respectively, within centrilobular and periportal regions. In livers of trans-stilbene oxide-pretreated rats, anti-epoxide hydrolase binding was increased by approximately 80% within both the midzonal and periportal regions and by approximately 43% within centrilobular regions. These immunohistochemical findings demonstrate that phenobarbital and trans-stilbene oxide both induce epoxide hydrolase nonuniformly within the liver lobule. However, while phenobarbital induces the enzyme to the greatest extent within midzonal hepatocytes and to the least extent within periportal hepatocytes, trans-stilbene oxide induces epoxide hydrolase equally within midzonal and periportal hepatocytes.

Sporidesmin toxicity in rabbits: Biochemical and morphological changes

The earliest lesion in rabbits dosed orally with 2 mg of sporidesmin per kg of body weight was necrosis of occasional hepatocytes 1 day after dosing. The most consistent lesion was a severe necrotizing cholangitis of medium and large-sized intrahepatic and extrahepatic bile ducts, first seen 2 days after dosing. Similar lesions were also present in the gall bladder of some rabbits. Expansion of portal triads with fibrous tissue and proliferating bile ductules progressed to pseudolobulation by 21 days. Other hepatic changes observed irregularly included large infarcts at the periphery of some lobes, and multiple small foci of coagulation necrosis in midzonal and periportal regions. Vascular necrosis and thrombosis, invariably adjacent to necrotic bile ducts, was presumably responsible for the hepatic necrosis. Serum cholesterol and total bilirubin concentrations and GGT activity reached peaks 15 days after dosing and were useful indicators of the severity of biliary lesions. Serum ID activity was the most useful indicator of hepatic necrosis following oral dosing with sporidesmin. The similarity between hepatobiliary lesions observed in sheep and rabbits with experimental sporidesmin toxicity suggests that the rabbit would be a useful model for studying methods of treatment and prevention of “facial eczema” in ruminants.

Identification and distribution of megakaryocyte colonies in murine spleen.

This study is the first report on the utilization of specific cell function to identify splenic megakaryocytic colonies. Stem cell differentiation into megakaryocytes was studied by injecting each irradiated murine syngeneic recipient with 1 x 10(6) spleen cells. Morphological identification of erythroid, granulocytic, megakaryocytic, and mixed and undifferentiated colonies was done by staining consecutive cryostat sections with hemotoxylin and eosin, benzidine, myeloperoxidase, and acetylcholinesterase. The variation in the distribution of hemopoietic colonies within the spleen was reflected in the different ratio values derived for erythroid, granulocytic, and megakaryocytic colonies at varying depth within the spleen. An increase by 50% of megakaryocyte colonies was seen within the splenic pulp in the midzone region, compared with the surface. This suggests a localized microenvironment conducive for megakaryocytopoiesis. The data emphasizes the importance of identifying colonies of all cell types in histological sections of the spleen and evaluating spleen sections at least at two levels, one adjacent to the surface and the other in the midzone area.

Toxicity of 3,3',4,4'- and 2,2',5,5'-tetrabromobiphenyl: correlation of activity with aryl hydrocarbon hydroxylase induction and lack of protection by antioxidants.

3,3',4,4'-Tetrabromobiphenyl is a minor component of commercial polybrominated biphenyl (PBB) mixture fireMaster BP-6 and is a potent inducer of aryl hydrocarbon hydroxylase (AHH). A single ip dose of 3,3',4,4'-tetrabromobiphenyl (150 mumol/kg) caused significant reduction in the growth rate in the immature male Wistar rat, as well as pale enlarged livers and marked reduction in thymus size. Under light microscopy, hepatocytes were enlarged and vacuolated. The vacuoles, which were most prominent in the midzonal region of the lobule, corresponded to fat droplets in oil-red-O-stained sections. The thymus, especially the cortex, was markedly depleted of lymphocytes. Neither the reduced growth, altered organ weights nor the histopathology was reversed for the duration of the study by the coadministration of the antioxidants butylated hydroxy anisole (BHA), butylated hydroxy toluene (BHT), or vitamin E. Vitamin E did, however counter the negative effect of 3,3',4,4'-tetrabromobiphenyl on growth during the first 5 d of the study. 2,2',5,5'-Tetrabromobiphenyl, also a minor component of fireMaster BP-6, is a weak phenobarbital-type inducer of cytochrome P-450. When administered at the same dose, 2,2',5,5'-tetrabromobiphenyl did not elicit any observed toxic effects. These data confirm the correlation between AHH induction and toxicity for these PBBs and suggest that 3,3',4,4'-tetrabromobiphenyl may significantly contribute to the toxicity of fireMaster BP-6. Although there is evidence that polychlorinated biphenyls, and perhaps 2,3,7,8-tetrachlorodibenzo-p-dioxin, exert certain toxic effects via a lipid peroxidation mechanism, the toxic changes measured during this study were not reversed by the administration of the antioxidants.

Immunohistochemical localization of glutathione S-transferases in livers of untreated rats.

Sheep antibodies raised against three isoenzymes of glutathione S-transferase (EC 2.5.1.18), transferases B, C, and E, which were isolated and purified to apparent homogeneity from rat liver, have been employed to localize these enzymes at the light microscopic level within livers of untreated rats. Using these antibodies in an unlabeled antibody peroxidase-antiperoxidase staining technique, each glutathione S-transferase was detected immunohistochemically within parenchymal cells throughout the liver lobule. In addition, immunohistochemical staining for transferases C and E, but not for transferase B, was observed within bile duct epithelium. While all parenchymal cells were stained with each glutathione S-transferase antibody, the patterns of immunohistochemical staining intensity observed across the liver lobule with the three anti-transferases were not uniform: parenchymal cells within the centrilobular region were more intensely stained for each lobular region were more intensely stained for each isoenzyme than were those within the midzonal and periportal regions of the lobule. The results of this immunohistochemical study thus demonstrate that glutathione S-transferases are not distributed uniformly throughout the liver lobule and that each transferase is present in the greatest concentration within the centrilobular region of the lobule.

Intracellular localization of human DNA polymerase alpha with monoclonal antibodies.

We have successfully established 16 stable murine hybridomas that secrete monoclonal antibodies specific for human DNA polymerase alpha. The results of immunocytochemical studies, using 4 of these monoclonal antibodies and immunoperoxidase detection methods, document the exclusively intranuclear localization of DNA polymerase alpha in three separate lines of cultured human cells. By light microscopy, the immunoperoxidase reaction product exhibits a diffuse pattern of distribution within the nucleoplasm, but nucleoli are clearly negative. In cultures of the transformed lines, KB nd BeWo, more than 955 of the cells are positive, suggesting that intranuclear DNA polymerase alpha antigens persist throughout the mitotic cycle. In striking contrast, in the normal diploid fibroblast line, WI-38, a smaller fraction of the cultured cells is positive, and there is no detectable polymerase alpha antigen in the closely apposed cells of microcolonies that are presumed to be contact-arrested and no longer mitotically cycling. In cells in mitosis that have dissolved their nuclear envelopes (and are thus transiently anucleate), the anti-polymerase alpha reaction continues to be strongly positive, and in this single circumstance the reaction product is diffusely distributed throughout the cell cytoplasm. Initial electron microscopic examination of KB cells confirms and extends these observations. The immunoperoxidase reaction product is essentially limited to the nuclear compartment and is predominantly distributed in the midzonal region of the nucleoplasm between centrally disposed nucleoli and peripherally located blocks of condensed chromatin.

Interalveolar pores in mouse lung. Regional distribution and alterations with age.

Quantitative analysis of the regional distribution of interalveolar pores in the lungs of BALB/c mice 1 to 28 months of age (n = 25) was performed by scanning electron microscopy. In all the age groups examined, the subpleural and peribronchiolar alveoli had significantly more pores than the midzonal region (p < 0.01). No significant changes were noted in the number of pores per alveolus between 3 and 26 months of age in the midzonal, peribronchiolar, and subpleural alveoli. We concluded that (1) intralevolar pores are regionally distributed in the lungs of BALB/c mice by 3 months of age, and (2) the number of pores do not significantly change with increasing age during the first 26 months.