Alcohol-Induced Lysosomal Damage and Suppression of Lysosome Biogenesis Contribute to Hepatotoxicity in HIV-Exposed Liver Cells.

Although the causes of hepatotoxicity among alcohol-abusing HIV patients are multifactorial, alcohol remains the least explored "second hit" for HIV-related hepatotoxicity. Here, we investigated whether metabolically derived acetaldehyde impairs lysosomes to enhance HIV-induced hepatotoxicity. We exposed Cytochrome P450 2E1 (CYP2E1)-expressing Huh 7.5 (also known as RLW) cells to an acetaldehyde-generating system (AGS) for 24 h. We then infected (or not) the cells with HIV-1ADA then exposed them again to AGS for another 48 h. Lysosome damage was assessed by galectin 3/LAMP1 co-localization and cathepsin leakage. Expression of lysosome biogenesis-transcription factor, TFEB, was measured by its protein levels and by in situ immunofluorescence. Exposure of cells to both AGS + HIV caused the greatest amount of lysosome leakage and its impaired lysosomal biogenesis, leading to intrinsic apoptosis. Furthermore, the movement of TFEB from cytosol to the nucleus via microtubules was impaired by AGS exposure. The latter impairment appeared to occur by acetylation of α-tubulin. Moreover, ZKSCAN3, a repressor of lysosome gene activation by TFEB, was amplified by AGS. Both these changes contributed to AGS-elicited disruption of lysosome biogenesis. Our findings indicate that metabolically generated acetaldehyde damages lysosomes and likely prevents their repair and restoration, thereby exacerbating HIV-induced hepatotoxicity.

Chronic ethanol consumption increases homocysteine accumulation in hepatocytes.

Results of previous studies have shown that chronic ethanol administration impairs methionine synthetase activity and decreases S-adenosylmethionine levels in the liver, indicating interference with homocysteine remethylation. The purpose of the present study was to investigate the effects of chronic ethanol feeding on the accumulation of homocysteine (Hcy), a potentially toxic agent. The research was divided into two experiments. In Experiment A, hepatocytes were isolated from pair-fed control and ethanol-fed rats after 2 weeks of feeding, and the release of Hcy into the medium was determined. Hepatocytes obtained from ethanol-fed rats released twice as much Hcy into the medium as did those obtained from controls. When hepatocytes were challenged by a methionine load, a marked increase in Hcy generation was observed, and the increase was further enhanced in hepatocytes obtained from ethanol-fed rats. In Experiment B, hepatocytes were isolated from pair-fed control and ethanol-fed rats after 4 weeks of feeding (the feeding time required for significant formation of alcoholic fatty liver in rats). In this experiment, similar results were obtained with Hcy generation as in Experiment A. In Experiment B, supplementation of the incubation medium with betaine prevented the increase in generation of Hcy by methionine-treated control cells as well as the generation of Hcy by cells of ethanol-treated rats. These results indicate that betaine may have the potential as a therapeutic agent against toxic Hcy formation.

Malondialdehyde-acetaldehyde-protein adducts increase secretion of chemokines by rat hepatic stellate cells.

Findings obtained from our recent studies have demonstrated that malondialdehyde, a product of lipid peroxidation, and acetaldehyde can react together with proteins in a synergistic manner and form hybrid protein conjugates, which have been designated as malondialdehyde-acetaldehyde (MAA)-protein adducts. These adducts have been detected in livers of ethanol-fed rats and are immunogenic because significant increases in circulating antibody titers against MAA-adducted proteins have been observed in ethanol-fed rats and more recently in human alcoholics. Although immunological factors may tend to perpetuate liver injury, little is known about the direct functional consequences of MAA-adducted proteins on the different cellular populations of the liver. Hepatic stellate cells (HSCs) have been shown to be pivotal in the pathogenesis of fibrosis and in the amplification and self-perpetuation of the inflammatory process. The present study was conducted to determine the effects of MAA-adducted proteins on the function of HSCs. Rat HSCs were exposed to various amounts of MAA-protein adducts and their unmodified controls, and the secretion of two chemokines, monocyte chemoattractant protein (MCP)-1 and macrophage inflammatory protein (MIP)-2, that are involved in the chemotaxis of monocytes/macrophages and neutrophils, respectively, was determined. We observed that bovine serum albumin-MAA induced a dose- and time-dependent increase in the secretion of both of these chemokines. These findings indicate that MAA-adducted proteins may play a role in the modulation of the hepatic inflammatory response and could contribute to the pathogenesis of alcoholic liver disease.

Malondialdehyde-acetaldehyde-adducted bovine serum albumin activates protein kinase C and stimulates interleukin-8 release in bovine bronchial epithelial cells.

Previous study results have demonstrated that cigarette smoke or acetaldehyde rapidly stimulates protein kinase C (PKC)-mediated release of interleukin-8 (IL-8) in bovine bronchial epithelial cells (BECs). Low concentrations of acetaldehyde combine synergistically with malondialdehyde to increase significantly maximal BEC PKC activity at 48 to 96 h stimulation. Because more than 95% of alcoholics are cigarette smokers, we hypothesized that malondialdehyde, an inflammation product of lipid peroxidation, and acetaldehyde, both a product of ethanol metabolism and a component of cigarette smoke, might stimulate PKC-mediated IL-8 release in BECs by malondialdehyde-acetaldehyde (MAA) adduct formation, rather than as free aldehydes. Protein kinase C activity is maximally elevated in BECs treated with 50 microg/ml of BSA-MAA from approximately 1 to 3 h. This activity subsequently begins to decrease by 4 to 6 h, with a return to baseline unstimulated kinase activity levels by 24 h. No activation of cyclic AMP-dependent protein kinase (PKA) or cyclic GMP-dependent protein kinase (PKG) was observed in BSA-MAA-treated BECs. The MAA adduct activation of PKC was followed by a fourfold to tenfold greater release of IL-8 over that observed for both BECs exposed to media only and BSA control-treated BECs. Protein kinase C activation and IL-8 release were blocked by pretreating BECs with 1 microM calphostin C or 100 nM of the PKC alpha-specific inhibitor, Go 6976. Isoform-specific inhibitors to PKC beta, PKC delta, and PKC zeta failed to inhibit completely MAA adduct-stimulated PKC or IL-8 release. Results of these studies indicate that metabolites derived from ethanol and cigarette smoke, such as acetaldehyde and malondialdehyde, form adducts that stimulate airway epithelial cell PKC alpha-mediated release of promigratory cytokines.

Ethanol feeding selectively impairs the spreading of rat perivenous hepatocytes on extracellular matrix substrates.

BACKGROUND: Hepatocytes require attachment and subsequent spreading on an extracellular matrix for their proper growth, function and survival. Our previous studies have shown that ethanol feeding selectively impairs perivenule hepatocyte attachment to various extracellular matrices. This study was undertaken to determine whether zonal differences in hepatocyte spreading in response to ethanol feeding occurs and to ascertain the influence of ethanol consumption on the zonal expression of the beta1 subunit of integrins, which are the major surface receptors responsible for matrix binding and subsequent interactions. METHODS: Hepatocytes from the perivenous and periportal regions of the liver were isolated by digitonin/collagenase perfusion from rats that were pair-fed for 2 to 3 weeks with a liquid diet containing either ethanol or isocaloric carbohydrate. The ability of perivenous and periportal hepatocytes to spread on plates coated with either type IV collagen, laminin, fibronectin or polylysine was determined. In addition, the isolated cells were used for the analysis of total cellular and surface beta1 integrin expression. RESULTS: With all of the matrix substrates tested, the spreading of perivenous hepatocytes isolated from the ethanol-fed animals was markedly impaired, while the spreading of periportal hepatocytes was essentially unaffected by ethanol feeding. Both the total cellular as well as the surface expression of the beta1 integrin subunit in perivenous cells from the ethanol-fed rats were significantly higher than from the perivenous control cells, whereas the total and surface expression of the beta1 integrin in periportal cells isolated from ethanol-fed and control rats were not significantly different. CONCLUSIONS: The results indicated that in addition to impairing hepatocyte attachment, ethanol feeding also impairs another critical step of the adhesion process, that of hepatocyte spreading on extracellular matrix substrates. This defect occurred preferentially in perivenous cells and not periportal cells and was associated with an increase in beta1 integrin expression, suggesting that a compensatory mechanism occurs as an attempt by the perivenous cells to overcome impaired cell-matrix interactions caused by ethanol. Overall, these alterations in extracellular matrix-hepatocyte interactions could lead to alterations of hepatocyte structure and function and potentially play a role in alcoholic liver injury.

Flow cytometric analysis of vesicular pH in rat hepatocytes after ethanol administration.

We examined the effect of ethanol administration on intravesicular pH in intact hepatocytes by applying a flow cytometric technique to detect fluorescein-isothiocyanate-dextran (FITC-dextran) in acidic vesicles. Rats were pair-fed liquid diets containing either ethanol or isocaloric carbohydrate for 1 to 5 weeks. Our study showed that ethanol administration increased the in situ pH of hepatic lysosomes by 0.15 to 0.2 pH units. This pH increase was sufficient to cause a significant reduction in lysosomal protein degradation. Long-term ethanol administration also caused a significant alkalinization of hepatic endosomes, and this increased pH was sustained over the course of vesicular acidification in hepatocytes incubated in vitro. Direct exposure of hepatocytes from rats fed control diet to either 25 mmol/L ethanol or 50 micromol/L colchicine also brought about a rapid alkalinization of acidic vesicles in a manner that resembled that seen in hepatocytes from ethanol-fed rats. These same treatments augmented the vesicular alkalinization already present in cells from ethanol-fed animals. Although ethanol administration had no effect on the content of the hepatic mannose-6-phosphate/IGFII receptor, the results indicate that sustained alkalinization of endosomes could have important functional consequences by impairing M-6-P/IGFII receptor recycling, thereby disrupting the delivery of newly synthesized hydrolases to lysosomes. This decreased complement of hydrolases within lysosomes together with alkalinization of the intralysosomal compartment would result in an overall decrease in lysosomal proteolysis.

Ethanol consumption alters trafficking of lysosomal enzymes and affects the processing of procathepsin L in rat liver.

In order to determine whether ethanol consumption alters the targeting of hepatic lysosomal enzymes to their organelles, we examined the sedimentation properties of lysosomal hydrolases in ethanol-fed rats and their pair-fed controls. Rats were fed a liquid diet containing either ethanol (36% of calories) or isocaloric maltose dextrin for one to five wk. Liver extracts were fractionated by Percoll density gradient centrifugation and fractions obtained were analyzed for the distribution of lysosomal marker enzymes. Heavy lysosomes were further purified from these gradients and the activity of specific hydrolases was determined. Compared with those from controls, isolated lysosomes from ethanol-fed rats showed a 20-50% reduction in the activity of lysosomal acid phosphatase and beta-galactosidase. Decreased intralysosomal hydrolase activity in ethanol-fed rats was associated with a significant redistribution of these enzymes as well as those of cathepsins B and L to lighter fractions of Percoll density gradients. This indicated an ethanol-elicited shift of these enzymes to lower density cellular compartments. In order to determine whether ethanol administration affects the synthesis and proteolytic maturation of hepatic procathepsin L, we conducted immunoblot analyses to quantify the steady-state levels of precursor and mature forms of cathepsin L in hepatic post-nuclear fractions. Ethanol administration caused a significant elevation in the steady-state level of the 39 kDa cathepsin L precursor relative to its 30 kDa intermediate and 25 kDa mature product. These results were confirmed by pulse-chase experiments using isolated hepatocytes exposed to [35S]methionine. Hepatocytes from both control and ethanol-fed rats incorporated equal levels of radioactivity into procathepsin L. However, during the chase period, the ratios of the 39 kDa procathepsin L to its 30 kDa intermediate and 25 kDa mature product in cells from ethanol-fed rats were 1.5-3-fold higher than those in controls. These results demonstrate that ethanol consumption caused a marked impairment in the processing of procathepsin L to mature enzyme, without affecting its synthesis. Taken together, our findings suggest that chronic ethanol consumption caused a deficiency in intralysosomal enzyme content by altering the trafficking and processing of these hydrolases into lysosomes.

Effects of ethanol administration on components of the ubiquitin proteolytic pathway in rat liver.

Hepatic protein accumulation during ethanol administration may result partly from an ethanol-elicited decline in hepatic protein degradation, which we have previously shown. We conducted the current studies to examine the effects of ethanol administration on the levels of hepatic ubiquitin, an 8.5-kd protein which is an important mediator of extralysosomal protein catabolism. Rats were pair-fed liquid diets containing either ethanol (36% of calories) or isocaloric maltose-dextrin for 1 to 5 weeks. Ubiquitin was immunochemically quantified by competitive enzyme-linked immunosorbent assay (ELISA) in crude cytosol fractions from whole liver and in 12,000g supernatants of hepatocyte lysates. Ubiquitin levels in hepatic cytosol fractions of ethanol-fed rats exceeded those of controls by about 30%. Isolated hepatocytes from ethanol-fed animals also showed a 40% to 75% elevation of ubiquitin above that in cells of pair-fed controls and this difference exceeded the relative rise in hepatocellular protein. In hepatocyte lysates subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, we detected monomeric ubiquitin and higher molecular mass ubiquitin-protein conjugates. However, the immunoblot analyses revealed no quantitative changes in the level of either free or conjugated ubiquitin. The ubiquitin conjugating activity of crude and diethyl aminoethyl-fractionated liver cytosols of ethanol-fed rats had equal capacities to those from controls in catalyzing the formation of ubiquitin-protein conjugates. Our findings indicate that chronic ethanol consumption increased the level of immunoreactive ubiquitin in rat liver. This may have resulted from enhanced ubiquitin production because of an ethanol-elicited stress response and/or decreased catabolism of ubiquitin and its conjugates. Our findings also provide no indication that the ethanol-elicited reduction in hepatic proteolysis is because of a ubiquitin-mediated mechanisms.

Ethanol consumption reduces the proteolytic capacity and protease activities of hepatic lysosomes.

Chronic ethanol consumption causes decreased hepatic protein degradation, resulting in protein accumulation within hepatocytes. In this investigation, we sought to determine whether chronic ethanol feeding alters the degradative capacity and protease activities of isolated hepatic lysosomes. Male Sprague-Dawley-derived rats were fed a liquid diet containing either ethanol (36% of calories) or isocaloric maltose-dextrin for 1-5 wk. Hepatic lysosomes were isolated by differential centrifugation and purified through Percoll gradients. Lysosomes obtained from livers of ethanol-fed rats degraded both endogenous protein substrates and the exogenously added radioactive substrate, 125I-RNase A, 26-42% more slowly than lysosomes from pair fed controls. The ethanol-elicited reduction in proteolytic capacity appeared to result in part, from a deficiency of the lysosomal cathepsins B, L, and H. Compared with controls, the specific activities of these enzymes were 31-45% lower in lysosomes from ethanol-fed rats. Immunoblot analyses also revealed that the intralysosomal as well as the intracellular content of cathepsin B was significantly lower in ethanol-fed rats. In contrast, ethanol consumption did not affect the cellular quantity of cathepsin L but lowered its amount in isolated lysosomes. Our findings suggest that chronic ethanol consumption causes a deficiency in lysosomal cathepsins by altering their biosynthesis and/or their trafficking into lysosomes.

Rat prostatic inhibin: less than 35 kDa protein(s).

An attempt was made to purify and physicochemically characterize the inhibin-like activity of rat ventral prostate based on the technique of immunosorption. It was possible to partially purify prostatic inhibin by affinity chromatography and the molecular size of inhibin-like material was shown to be less than 35 kDa.

A correlative study of in vivo and in vitro labeling index using bromodeoxyuridine in human brain tumors.

The labeling index (LI) of 216 cases of human brain tumors was determined by the immunohistochemical technique with monoclonal antibody to bromodeoxyuridine (BrdU). The proliferative potential of 110 cases was estimated using the intra-operative intravenous infusion of BrdU at a dose of 200 mg/sq m. In another 106 cases, the in vitro technique of incubating freshly resected tumor tissue fragments with 100 microM bromodeoxyuridine was used. The BrdU LI in these tumors was then correlated with the histological types and the data as determined by both the in vivo and in vitro BrdU incorporation were compared. The results indicate that although in vivo and in vitro techniques could possibly provide equivalent data in some histologic types, a clear statistically valid proof however is not apparent from this study.

Ethanol administration alters the proteolytic activity of hepatic lysosomes.

Protein accumulation in liver cells contributes to alcohol-induced hepatomegaly and is the result of an ethanol-elicited deceleration of protein catabolism (Alcohol Clin Exp Res 13:49, 1989). Because lysosomes are active in the degradation of most hepatic proteins, the present studies were conducted to determine whether ethanol administration altered the proteolytic activities of partially purified hepatic lysosomes. Rats were fed liquid diets containing either ethanol (36% of calories) or isocaloric maltodextrin for periods of 2-34 days. Prior to death, all animals were injected with [3H]leucine to label hepatic proteins. Rats subjected to even brief periods of ethanol feeding (2-8 days) exhibited significant hepatomegaly and hepatic protein accumulation compared with pair-fed control animals. Crude liver homogenates and isolated lysosomal-mitochondrial and cytosolic subfractions were incubated at 37 degrees C, and the acid-soluble radioactivity generated during incubation was measured as an index of proteolysis. At neutral pH, in vitro protein breakdown in incubated liver homogenates and subcellular fractions from control and ethanol-fed rats did not differ significantly. The extent of protein hydrolysis increased when samples were incubated at pH 5.5, which approximates the pH optimum for catalysis by lysosomal acid proteases. Under the latter conditions, partially purified lysosomes from control animals had 2-fold higher levels of proteolysis than corresponding fractions from ethanol-fed rats. The difference in proteolytic capacity appeared to be related to a lower latency and a higher degree of fragility of lysosomes from ethanol-fed rats at the acidic pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Primary cerebellopontine angle choroid plexus papilloma: a case report and review of literature.

A case of primary choroid plexus papilloma of the cerebellopontine (CP) angle is described in a 28 years old man. He presented with hearing loss, right facial palsy and spastic quadriparesis (4/5). He also had markedly increased intracranial pressure. CT scan revealed a large high attenuating lesion in right CP angle with gross hydrocephalus. The patient was operated with the clinical and radiological diagnosis of right sided acoustic tumor with brainstem compression. Radical tumour excision was performed, seven days following VP shunt. Patient had immediate postoperative deterioration followed by a steady recovery. The possibility of a secretory choroid plexus papilloma is discussed.

Cell culture studies on human nerve sheath tumors.

The main controversy about nerve sheath tumors (NSTs) has been their histogenesis. A Schwann cell origin has been proposed by many investigators for both schwannomas and neurofibromas. However Erlandson and Woodruff observed that while schwannomas appeared to be composed predominantly of Schwann cells, neurofibromas consisted of mainly perineurial cells. In addition, variable numbers of fibroblast-like cells and intermediate cells also have been reported in the 2 lesions. Whether these represent distinct cell types or variants of Schwann cells is still debatable. In an attempt to solve this controversy, the present study was undertaken to observe the morphology and the behaviour of these tumors in culture. These studies showed that all nerve sheath tumors are basically of Schwann cell origin and that intermediate cells and fibroblasts are variants of Schwann cell. Tissue culture studies done chiefly on schwannomas showed that the morphological features of schwannomas are preserved in 'in vitro culture' condition and therefore the difference between neurofibroma and schwannoma appears to be due to inherent differentiating property of the Schwann cells along with some environmental stimulus.

A sequential cell kinetic study of meningioma cells in primary explant culture using bromodeoxyuridine.

The present study was undertaken to evaluate the sequential BrdU-LI at weekly intervals upto four weeks in 18 primary explant cultures of meningiomas. This revealed three distinct patterns of growth which could be arbitrarily defined as 'degenerating' (group I), 'proliferating' (group II) and 'adaptive' (group III) types. Interestingly two cases of malignant and two of recurrent meningiomas fell into the 'degenerating' group I pattern. The possible explanations for the observed relatively higher in vitro LI values compared to lower in vivo values as reported in the literature and the theoretical implications of the three distinct patterns of sequential LI values are discussed.

A transmission and scanning electron microscopic study of tumoral and peritumoral microblood vessels in human gliomas.

The tumor microblood vessels (MBVs) of 25 cases of gliomas of varying grades were studied and compared with those in peritumoral region using both transmission and scanning electron microscopy (TEM and SEM). The TEM study revealed numerous villous projections with pinocytotic vesicles (PCVs) and large vacuoles (LVs) concentrated mainly at the luminal aspect in tumor MBVs which increased with increasing severity of edema. The peritumoral MBVs, in addition to showing some increase in villous projections on the luminal surface, also showed increased number of PCVs and LVs concentrated at the abluminal aspect with some of them even communicating with the extravascular space. The SEM study largely corroborated the TEM findings. The sites of formation of PCVs and LVs appeared as small pits or large craters on the luminal surface of the endothelial cells of tumor MBVs. We feel that the morphological evidence of increased permeability in tumor MBVs represents their role in the development of edema and that the occurrence of reverse pinocytosis in peritumoral MBVs is a distinct possibility which may be associated with resorption of edema fluid.

In-vivo proliferative potential of primary human brain tumors; its correlation with histological classification and morphological features: I. Gliomas.

This study was undertaken to investigate in-vivo proliferative potential of neoplastic cells in 66 cases of gliomas of different histological types following peroperative intravenous infusion of bromodeoxyuridine (BrdU). Histological typing according to the recent modification of WHO classification did not often correlate with in-vivo cell kinetics. Among the different morphological features used for the classification, necrosis, mitosis, increased cell density and increased endothelial cell proliferation showed good correlation with tumor cell labelling index (LI) (p < 0.01-p < 0.001). A preliminary follow-up study of 36 cases for a period ranging from 9 to 36 mths suggested the possibility that higher in-vivo tumor cell LI might be associated with an early recurrence. Thus in vivo BrdU LI may supplement the histological classification of gliomas and together they may help in a better assessment of their growth rate, degree of malignancy and biological behaviour which in turn facilitate the planning of therapeutic management for individual cases.

Morphological appearance, growth kinetics and glial fibrillary acidic protein (GFAP) expression in primary in vitro explant culture of astrocytic neoplasms.

Astrocytomas of different grades of malignancy were cultured as primary explant and their sequential growth pattern, glial fibrillary acidic protein (GFAP) expression and labelling index (LI) using bromodeoxyuridine (BrdU) were assessed and correlated with the grade of malignancy of the original tumor tissue. Low-grade astrocytomas showed patterns of growth that diverged from anaplastic astrocytomas and glioblastoma multiforme. The GFAP expression decreased with increasing time in culture in all astrocytomas irrespective of the grading. Maximum GFAP was, however, expressed in the morphologically well-differentiated stellate cells. Contrary to expectations, lower BrdU LI was observed in glioblastoma multiforme in comparison to low-grade astrocytomas, which suggests some unidentified mechanism of differentiation in vitro for astrocytomas of higher grade of malignancy. Hence, in contrast to reported literature on the prognostic value of studies on primary cultures, the present study cautions the extrapolation of the in vitro findings for astrocytomas.

In-vivo proliferative potential of primary human brain tumors; its correlation with histological classification and morphological features: II. Nonglial tumors.

In-vivo cell kinetics study following peroperative intravenous infusion of bromodeoxyuridine (BrdU) was done in 10 cases of primitive neuroectodermal tumors (PNET) and 44 nonglial tumors of different histological types. The histological features usually regarded as indicators of aggressive behaviour were examined in these tumors and correlated with in-vivo labelling index (LI). In the case of meningiomas, increased cellularity, pleomorphism and mitosis had no correlation with LI. Benign non-recurrent meningiomas usually showed LI < or = 1% (mean 0.6 +/- 0.3%) whereas recurrent and malignant meningioma showed higher LI (mean 2.6 +/- 0.5% and 2.8 +/- 0.4% respectively). Follow-up study suggested that meningiomas having benign histological appearance with LI > 1% might have increased chance of recurrence. In cases of PNETs among different histological features, mitosis and differentiation seemed to be related to the biological behaviour. Astrocytic differentiation was associated with lower rate of proliferation. In pituitary adenomas different hormone producing and null cell adenomas showed similar low proliferative potential (0.6 +/- 0.3%). Benign nerve sheath tumors, craniopharyngioma and choroid plexus papilloma showed low in-vivo LI of less than 1%. Thus the present study revealed the inadequacies of routine histological examination in assessing the aggressiveness of the nonglial tumors, especially meningiomas. In-vivo LI may be a good supplement to histological diagnosis as well as helping to assess the prognosis and accordingly the management of individual cases.

Ultrastructural study of mixed growth hormone & prolactin secreting pituitary adenomas.

An ultrastructural study was done on 15 mixed growth hormone (GH) and prolactin (PRL)-secreting pituitary adenomas surgically removed from acromegalic patients with hyper-prolactinaemia, in order to see whether the 2 hormones were present in the same cell or in different cells. Double labelling immunogold technique was used for simultaneous ultrastructural localization of GH and PRL. It was found that each neoplastic cell in these 15 tumours (30 to 50 cells were studied in each case) contained 4 populations of granules viz., (i) granules positive for only GH; (ii) granules positive for only PRL; (iii) granules positive for both GH and PRL; and (iv) granules negative for both GH and PRL (unlabelled). Though the relative percentage of these 4 types of granules varied from cell to cell even within the same tumour, the major population (49.9 to 96%) was constituted by the mixed granules showing labelling for both GH and PRL. Almost all the cells examined from each tumour appeared to be mammosomatotrophs. Thus, the study indicated that mammosomatotroph adenomas are perhaps more common among mixed GH and PRL--secreting pituitary adenomas than previously believed. It could be important to recognize these tumours from the therapeutic point of view.