Effects of curcumin on cancer cell mitochondrial function and potential monitoring with ¹8F-FDG uptake.

A better understanding of how curcumin influences cancer cell biology could help devise new strategies to enhance its antitumor effect. Many curcumin actions are proposed to occur by targeting mitochondrial function, among which glucose metabolism and reactive oxygen species (ROS) production are pivotal. However, little is known of how curcumin influences cancer cell glucose metabolism. We thus evaluated the effect of curcumin on cancer cell glucose metabolism and mitochondrial function, and further investigated whether these responses could be modified to enhance the anticancer potency of the compound. MCF-7 breast cancer cells treated with curcumin were measured for 18F-fluorodeoxyglucose (18F­FDG) uptake, lactate production, hexokinase activity, oxygen consumption rate (OCR), ROS production and mitochondrial membrane potential (MMP). Activation of signaling pathways was evaluated by western blots, and cell survival was assessed with sulforhodamine B assays. Curcumin stimulated a 3.6-fold increase of 18F-FDG uptake in MCF-7 cells, along with augmented hexokinase activity and lactate efflux. This was accompanied by significantly suppressed cellular OCR, consistent with a metabolic shift to glycolytic flux. Inhibiting this metabolic response with 2-deoxyglucose (2-DG) blocked curcumin-induced mTOR activation and resulted in a greater anti-proliferative effect. Curcumin-induced MMP depolarization led to reduced ROS production, which may hinder the anticancer effect of the compound. Intracellular ROS was completely restored by adding Cu2+, which can bind and modify the curcumin's physico-chemical property, and this resulted in a marked potentiation of its anti-proliferative effect. Thus, curcumin suppresses cancer cell MMP and ROS generation, and this response is accompanied by stimulated 18F-FDG uptake via shifting of metabolism from mitochondrial respiration to glycolytic flux. These mitochondrial and metabolic responses may provide potential targets that can help enhance the anticancer action of curcumin.

Role of choline deficiency in the Fatty liver phenotype of mice fed a low protein, very low carbohydrate ketogenic diet.

Though widely employed for clinical intervention in obesity, metabolic syndrome, seizure disorders and other neurodegenerative diseases, the mechanisms through which low carbohydrate ketogenic diets exert their ameliorative effects still remain to be elucidated. Rodent models have been used to identify the metabolic and physiologic alterations provoked by ketogenic diets. A commonly used rodent ketogenic diet (Bio-Serv F3666) that is very high in fat (~94% kcal), very low in carbohydrate (~1% kcal), low in protein (~5% kcal), and choline restricted (~300 mg/kg) provokes robust ketosis and weight loss in mice, but through unknown mechanisms, also causes significant hepatic steatosis, inflammation, and cellular injury. To understand the independent and synergistic roles of protein restriction and choline deficiency on the pleiotropic effects of rodent ketogenic diets, we studied four custom diets that differ only in protein (5% kcal vs. 10% kcal) and choline contents (300 mg/kg vs. 5 g/kg). C57BL/6J mice maintained on the two 5% kcal protein diets induced the most significant ketoses, which was only partially diminished by choline replacement. Choline restriction in the setting of 10% kcal protein also caused moderate ketosis and hepatic fat accumulation, which were again attenuated when choline was replete. Key effects of the 5% kcal protein diet - weight loss, hepatic fat accumulation, and mitochondrial ultrastructural disarray and bioenergetic dysfunction - were mitigated by choline repletion. These studies indicate that synergistic effects of protein restriction and choline deficiency influence integrated metabolism and hepatic pathology in mice when nutritional fat content is very high, and support the consideration of dietary choline content in ketogenic diet studies in rodents to limit hepatic mitochondrial dysfunction and fat accumulation.

The biarylpyrazole compound AM251 alters mitochondrial physiology via proteolytic degradation of ERRα.

The orphan nuclear receptor estrogen-related receptor alpha (ERRα) directs the transcription of nuclear genes involved in energy homeostasis control and the regulation of mitochondrial mass and function. A crucial role for controlling ERRα-mediated target gene expression has been ascribed to the biarylpyrazole compound 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide (AM251) through direct binding to and destabilization of ERRα protein. Here, we provide evidence that structurally related AM251 analogs also have negative impacts on ERRα protein levels in a cell-type-dependent manner while having no deleterious actions on ERRγ. We show that these off-target cellular effects of AM251 are mediated by proteasomal degradation of nuclear ERRα. Cell treatment with the nuclear export inhibitor leptomycin B did not prevent AM251-induced destabilization of ERRα protein, whereas proteasome inhibition with MG132 stabilized and maintained its DNA-binding function, indicative of ERRα being a target of nuclear proteasomal complexes. NativePAGE analysis revealed that ERRα formed a ∼220-kDa multiprotein nuclear complex that was devoid of ERRγ and the coregulator peroxisome proliferator-activated receptor γ coactivator-1. AM251 induced SUMO-2,3 incorporation in ERRα in conjunction with increased protein kinase C activity, whose activation by phorbol ester also promoted ERRα protein loss. Down-regulation of ERRα by AM251 or small interfering RNA led to increased mitochondria biogenesis while negatively impacting mitochondrial membrane potential. These results reveal a novel molecular mechanism by which AM251 and related compounds alter mitochondrial physiology through destabilization of ERRα.

Radiolabeled Cu-ATSM as a novel indicator of overreduced intracellular state due to mitochondrial dysfunction: studies with mitochondrial DNA-less ρ0 cells and cybrids carrying MELAS mitochondrial DNA mutation.

OBJECTIVES: Radiolabeled Cu-diacetyl-bis (N(4)-methylthiosemicarbazone) (*Cu-ATSM), including (60/62/64)Cu-ATSM, is a potential imaging agent of hypoxic tumors for positron emission tomography (PET). We have reported that *Cu-ATSM is trapped in tumor cells under intracellular overreduced states, e.g., hypoxia. Here we evaluated *Cu-ATSM as an indicator of intracellular overreduced states in mitochondrial disorders using cell lines with mitochondrial dysfunction. METHODS: Mitochondrial DNA-less ρ(0)206 cells; the parental 143B human osteosarcoma cells; the cybrids carrying mutated mitochondria from a patient of mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) (2SD); and that carrying wild-type one (2SA) were used. Cells were treated under normoxia or hypoxia, and (64)Cu-ATSM uptake was examined to compare it with levels of biological reductant NADH and NADPH. RESULTS: ρ(0)206 cells showed higher (64)Cu-ATSM uptake than control 143B cells under normoxia, whereas (64)Cu-ATSM uptake was not significantly increased under hypoxia in ρ(0)206 cells. Additionally, (64)Cu-ATSM uptake showed correlate change to the NADH and NADPH levels, but not oxygenic conditions. 2SD cells showed increased (64)Cu-ATSM uptake under normoxia as compared with the control 2SA, and (64)Cu-ATSM uptake followed NADH and NADPH levels, but not oxygenic conditions. CONCLUSIONS: (64)Cu-ATSM accumulated in cells with overreduced states due to mitochondrial dysfunction, even under normoxia. We recently reported that (62)Cu-ATSM-PET can visualize stroke-like episodes maintaining oxygen supply in MELAS patients. Taken together, our data indicate that *Cu-ATSM uptake reflects overreduced intracellular states, despite oxygenic conditions; thus, *Cu-ATSM would be a promising marker of intracellular overreduced states for disorders with mitochondrial dysfunction, such as MELAS, Parkinson's disease and Alzheimer's disease.

Evaluation of 64Cu-labeled acridinium cation: a PET radiotracer targeting tumor mitochondria.

This report presents the synthesis and evaluation of (64)Cu(DO3A-xy-ACR) (DO3A-xy-ACR = 2,6-bis(dimethylamino)-10-(4-((4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)methyl)benzyl)acridin-10-ium) as a radiotracer for imaging tumors in athymic nude mice bearing U87MG glioma xenografts by PET (positron emission tomography). The biodistribution data suggested that (64)Cu(DO3A-xy-ACR) was excreted mainly through the renal system with >65% of injected radioactivity being recovered from urine samples at 1 h postinjection (p.i.). The tumor uptake of (64)Cu(DO3A-xy-ACR) was 1.07 ± 0.23, 1.58 ± 0.55, 2.71 ± 0.66, 3.47 ± 1.19, and 3.52 ± 1.72%ID/g at 0.5, 1, 2, 4, and 24 h p.i., respectively. (64)Cu(DO3A-xy-ACR) had very high liver uptake (31.90 ± 3.98, 24.95 ± 5.64, 15.20 ± 4.29, 14.09 ± 6.82, and 8.18 ± 1.27%ID/g at 0.5, 1, 2, 4, and 24 h p.i., respectively) with low tumor/liver ratios. MicroPET studies showed that the tumors were clearly visualized as early as 30 min p.i. in the glioma-bearing mouse administered with (64)Cu(DO3A-xy-ACR). The high liver radioactivity accumulation was also seen. (64)Cu(DO3A-xy-ACR) had a relatively high metabolic stability during excretion via both renal and hepatobiliary routes, but it was completely decomposed in the liver homogenate. We explored the localization mechanism of Cu(DO3A-xy-ACR) using both U87MG human glioma and the cultured primary U87MG glioma cells. The results from the cellular staining assays showed that (64)Cu(DO3A-xy-ACR) is able to localize in the mitochondria of living U87MG glioma cells due to the enhanced negative mitochondrial potential as compared to normal cells. Although (64)Cu(DO3A-xy-ACR) is not an ideal PET radiotracer for tumor imaging due to its high liver uptake, the results from this study strongly suggest that (64)Cu-labeled acridinium cations are indeed able to localize in the energized mitochondria of tumor cells.

Tracking cellular stress with labeled FMAU reflects changes in mitochondrial TK2.

PURPOSE: Fluoropyrimidines like 1-(2'-deoxy-2'-fluoro-beta-D: -arabinofuranosyl)-thymine (FMAU) and 3'-deoxy-3'-fluorothymidine (FLT) accumulate in tumors and are being used as positron emission tomography tumor-imaging tracers. Proliferating tissues with high thymidine kinase 1 (TK1) activity retain FLT; however, the mechanism of selective accumulation of FMAU in tumors and certain other tissues requires further study. METHODS: Retention of [(3)H]FLT and [(3)H]FMAU was measured in prostate cancer cell lines PC3, LNCaP, DU145, and the breast cancer cell line MD-MBA-231, and the tracer metabolites were analyzed by high-performance liquid chromatography (HPLC). FMAU retention, thymidine kinase 2 (TK2) activity, and mitochondrial mass were determined in cells stressed by depleted cell culture medium or by treating with oxidative, reductive, and energy stress, or specific adenosine monophosphate-activated protein kinase activator, or eIF2 inhibitor. TK1 and TK2 activities and mitochondrial mass were determined by FLT phosphorylation, 1-beta-D: -arabinofuranosylthymine (Ara-T) phosphorylation, and flow cytometry, respectively. RESULTS: FMAU retention in rapidly proliferating cancer cell lines was five to ten times lower than FLT after 10 min incubation. HPLC analysis of the cellular extracts showed that phosphorylated tracers are the main retained metabolites. Nutritional stress decreased TK1 activity and FLT retention but increased retained FMAU. TK2 inhibition decreased FMAU retention and phosphorylation with negligible effects on FLT. Oxidative, reductive, or energy stress increased FMAU retention and correlated with mitochondrial mass (r (2) = 0.88, p = 0.006). FMAU phosphorylation correlated with increased TK2 activity (r (2) = 0.87, p = 0.0002). CONCLUSION: FMAU is preferably phosphorylated by TK2 and can track TK2 activity and mitochondrial mass in cellular stress. FMAU may provide an early marker of treatment effects.

[Mitochondrial damage in human sperm caused by the antineoplastic agent betulinic acid]. / Mitochondriale Schädigung von Humanspermien durch das Onkotherapeutikum Betulinsäure.

BACKGROUND AND OBJECTIVE: Human spermatozoa post-ejaculation show all elements of intrinsic (via mitochondria) and extrinsic (via death receptors) programmed cell death or apoptosis. One experimental therapeutic agent for malignant melanoma is betulinic acid (BA), a cytotoxic agent which induces intrinsic apoptosis via direct effects on mitochondria. To assess the potential side effects of systemic BA, its effects on motility, mitochondrial transmembrane potential (mTMP) and the apoptotic enzymes caspase-9 and -3, were monitored in human ejaculated spermatozoa. PATIENTS AND METHODS: Semen samples from 33 healthy volunteers were examined after incubation with 60 microg/mL betulinic acid for 5 and 60 minutes. RESULTS: Treatment with betulinic acid caused an immediate disruption of mitochondrial transmembrane potential and activation of the "death enzymes" caspase-9 and -3. The loss of mitochondrial potential was accompanied by a significant decrease of spermatozoal motility. CONCLUSION: Our results suggest that inducers of the mitochondrial pathway of apoptosis used in the treatment of malignant melanoma damage the sensitive mitochondria of spermatozoa.

Monitoring mitochondrial metabolisms in irradiated human cancer cells with (99m)Tc-MIBI.

Cationic hexakis(2-methoxyisobutylisonitrile)-technetium-99m ((99m)Tc-MIBI), an agent for scintigraphic detection and imaging of tumors, accumulates in mitochondria of various cells and tissues of high mitochondrial metabolic activity. To monitor the mitochondrial metabolisms of human cancer cells exposed to ionizing radiation, uptake of (99m)Tc-MIBI in an irradiated human lung cancer cell line (A549) was measured at 1-12 h following 0-9 Gy irradiation in vitro. Mitochondrial membrane potential, an index of mitochondrial activity, was also determined by flow cytometry with 3,3'-dihexyloxacarbocyanine (DiOC6(3)). At 1 h after 3 and 9 Gy irradiation, cellular (99m)Tc-MIBI accumulation increased by 10.5 +/- 1.6 and 16.8 +/- 5.6% compared with controls, respectively (P < 0.01) DiOC6(3) measurement also showed increased mitochondrial membrane potentials immediately after irradiation, consistent with (99m)Tc-MIBI changes. The present findings showed that the transient hyperactivated mitochondrial metabolism and subsequently decreased activities following irradiation were monitored by determining the cellular (99m)Tc-MIBI accumulation, suggesting the possibility of (99m)Tc-MIBI scintigraphy as a functional imaging to monitor tumor metabolisms after radiation therapy.

Biological factors influencing parathyroid localization.

The major factor influencing scintigraphic detection of abnormal parathyroid glands seems to be their size. However, false-negative results have been reported in large glands while some very small adenomas have been identified. Other factors can influence 99mTc-MIBI and 99mTc-Tetrofosmin uptake and therefore the accurate detection of hyperfunctioning glands depends also on these. Increases in both perfusion and functional activity and targeting of abundant mitochondria-rich oxyphil cells seem to be relevant mechanisms of uptake. A relationship has been observed between the intensity of focal uptake in the parathyroid glands and the cell cycle phases for patients with secondary hyperparathyroidism. Higher uptake grades correlated with the active growing phase, showing that scintigraphy accurately reflects the functional status of the hyperplastic parathyroid glands. Serum calcium levels may modify radiotracer kinetics by influencing the membrane potential. In addition, P-glycoprotein or multidrug resistance (MDR) associated protein expression may play an important role in the false-negative results of parathyroid scintigraphy. If the lipophilic cationic radiotracers used in parathyroid scintigraphy are transported by the same mechanism as the anticancer drugs, they will be quickly eliminated from the parathyroid glands containing P-glycoprotein or MDR-related protein expression and the uptake in images will be negative. In parathyroid glands with no P-glycoprotein or MDR-related protein expression, the radiotracers remain in the cells, making it easier to detect them by scintigraphy.

Ultrasonic low-energy treatment: a novel approach to induce apoptosis in human leukemic cells.

OBJECTIVE: We evaluated the cytotoxic effect of ultrasonic irradiation at low energy on the viability of normal and leukemic cells and the potential mechanisms of action inducing this cytotoxicity. MATERIALS AND METHODS: Human leukemia cell lines (K562, HL-60, KG1a, and Nalm-6), primary leukemic cells, and normal mononuclear cells are treated by ultrasound at a frequency of 1.8 MHz during various exposure times (acoustical power of 7 mW/mL) and immediately tested for cell viability by the trypan blue exclusion assay. Apoptosis is evaluated by cell morphology, phosphatidylserine exposure, and DNA fragmentation. The mitochondrial potential, glutathione content, caspase-3 activation, PARP cleavage, and bcl-2/bax ratio are tested by flow cytometry. Cloning efficiency is evaluated by assays in methylcellulose. RESULTS: The technique we describe here, using minute amounts of energy and in the absence of any chemical synergy, specifically triggers apoptosis in leukemic cells while necrosis is significantly reduced. Ultrasonic treatment of 20 seconds' duration induces a series of successive phases showing the characteristic features of apoptosis: mitochondrial transmembrane potential disturbances, loss of phosphatidylserine asymmetry, morphological changes, and, finally, DNA fragmentation. In contrast to K562 cells, for which a significant reduction of cloning efficiency is observed, the growth of hematopoietic progenitors is totally unaffected. Ultrasound treatment is also associated with depletion of cellular glutathione content, suggesting a link with the oxidative stress. Moreover, the fact that active oxygen scavengers reduce ultrasonic-induced apoptosis suggests a sonochemical mechanism. CONCLUSION: The cell damage observed after exposure of leukemic cells to ultrasound is associated with the apoptotic process and may be a promising tool for a smooth, specific, and effective ex vivo purging of leukemic cells.

Mitochondrial uptake of sestamibi distinguishes between normal, inflammatory breast changes, pre-cancers, and infiltrating breast cancer.

The evaluation of breast tissue using nuclear imaging is dependent upon the delivery and uptake of the isotope by breast tissue. This is dependent upon blood flow to the breast and functioning mitochondria. This 2-part study investigated (1) differences in uptake of sestamibi when blood flow is enhanced (breast enhanced scintigraphy test [BEST]), and (2) differences in isotope uptake in normal (Nl) breast tissue, inflammatory changes in breast tissue (ICB), and breast cancer (CA). In the first part of the study, 10 women were compared using both Miraluma and BEST imaging; in the second part, 195 people were studied using BEST imaging only. The results were compared with histopathologic specimens. Little difference was noted between Miraluma and BEST imaging in the first part. Women with ICB showed a statistically significant (P <.05) increase in isotope uptake using BEST imaging. This difference was even more significant (P <.005) in women with CA. During the second part of the study, BEST imaging demonstrated an exponential increase in tracer uptake. When maximal count activity was compared, there was a statistically significant (P <.001) difference between Nl and ICB, between ICB and atypia (A), and between A and CA. BEST imaging demonstrated significant increases in isotope delivery when compared with Miraluma imaging. These differences allowed differentiation of breast tissue, including the detection of early changes in breast tissue.

Nitric oxide prevents intestinal mitochondrial dysfunction induced by surgical stress.

BACKGROUND: The intestine is highly susceptible to free radical-induced damage and earlier work has shown that surgical stress induces generation of oxygen free radicals in enterocytes, resulting in intestinal damage along with changes in mitochondrial structure and function. Nitric oxide is an important mediator of gastrointestinal function and this study looked at the effect of nitric oxide on surgical stress-induced intestinal mitochondrial alterations. METHODS: Controls and rats pretreated with the nitric oxide donor L-arginine were subjected to surgical stress by opening the abdominal wall and handling the intestine. Enterocytes were isolated, mitochondria prepared and the protection offered by L-arginine against damage due to surgical stress was determined. Protection to structural as well as functional aspects of mitochondria was examined. RESULTS: Mild handling of the intestine affected the enterocyte mitochondrial structure as assessed by lipid composition and electron microscopy. Mitochondria were also functionally impaired with altered calcium flux and decreased respiratory control ratio. Pretreatment with the nitric oxide synthase substrate L-arginine prevented these damaging effects of surgical stress. Protection with arginine was abolished by the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester, indicating the role of nitric oxide. CONCLUSION: Surgical stress in the small intestine can affect enterocyte mitochondrial structure and function. These damaging effects can be prevented by nitric oxide, an important modulator of cellular function.

Cephaloridine-induced inhibition of cytochrome c oxidase activity in the mitochondria of cultured renal epithelial cells (LLC-PK(1)) as a possible mechanism of its nephrotoxicity.

To clarify the mechanism of cephalosporin nephrotoxicity, the effects of cephaloridine (CLD), a nephrotoxic cephalosporin antibiotic, on the mitochondria of the pig kidney proximal tubular epithelial cell line LLC-PK(1) were studied in culture. The activity of cytochrome c oxidase in the mitochondria of LLC-PK(1) cells was significantly decreased from 9 h after addition of 1.0 mM CLD to the cultured cells. These effects were dose-dependent and accompanied with a significant decrease in the ATP content in the cells, followed by marked morphological changes in the mitochondria. These alterations were observed in the treated cells before the increase in lipid peroxidation. The activities of NADH-cytochrome c reductase and succinate dehydrogenase in the mitochondria and NADPH-cytochrome P450 reductase, NADH-cytochrome b(5) reductase, and 7-ethoxycoumarin O-deethylase in the microsomes of the treated cells were not affected. Superoxide anion production by the mitochondria prepared from LLC-PK(1) cells or NADH-cytochrome c reductase was not affected by addition of CLD (1-10 mM), but adriamycin (0.1 mM) or paraquat (0.1 mM) significantly increased the superoxide anion production. These results suggested that the primary action of CLD is inhibition of cytochrome c oxidase activity in the mitochondrial electron transport chain, which decreases intracellular ATP content in renal tubular epithelial cells and that these effects of CLD are followed by increased lipid peroxidation and cellular injury.

Effects of depletion of mitochondrial DNA in metabolism secretion coupling in INS-1 cells.

Mitochondrial dysfunction due to alterations in the mitochondrial genome (mtDNA) has recently attracted much attention, with the finding that mutations in the mitochondrially encoded proteins perturb cell function. Several disorders have been linked to such genetic changes, including a specific diabetic phenotype. Using ethidium bromide (EtBr) that intercalates into mtDNA, we have effectively eliminated functions under the control of mtDNA from the highly differentiated INS-1 insulin-secreting cell line. We have investigated the consequences on insulin secretion, mitochondrial enzyme activity, organelle structure, and membrane polarization in such cells (INS-1 rho0). Under these conditions, the mitochondrial membrane potential fails to hyperpolarize in response to either glucose or methylsuccinate. In agreement with this finding, the morphology of the mitochondria is altered in the presence of EtBr, sharing similarities with mitochondria in which the membrane potential has been collapsed with the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). In addition, there is no effect of either nutrient secretagogue at the level of the plasma membrane potential, although the effect of the depolarizing agent KCl on membrane depolarization is completely preserved. Similarly, glucose and methylsuccinate fail to increase insulin secretion, whereas KCl is still effective. To test further the effects of mtDNA depletion on exocytosis, we permeabilized INS-1 cells with Staphylococcus aureus alpha-toxin, which forms small holes in the plasma membrane. In contrast to control cells, mitochondrial substrates were incapable of stimulating insulin secretion in mtDNA-deficient cells, emphasizing that the defect in secretion lies at the level of mitochondrial function rather than in the exocytotic process. The results indicate the paramount importance of the mitochondria in the downstream effects elicited by exposure to elevated concentrations of nutrient secretagogue.

Differential mechanism of retention of Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (Cu-PTSM) by brain and tumor: a novel radiopharmaceutical for positron emission tomography imaging.

The reductive retention of 62Cu-PTSM was comparatively studied in the brain and Ehrlich ascites tumor cells by electron spin resonance spectrometry and nonradioactive Cu-PTSM. In the brain, only the mitochondrial fraction showed the ability to reduce Cu-PTSM, and the other subcellular fractions did not. In contrast, the cytosolic fraction of Ehrlich ascites tumor cells was the specific site of Cu-PTSM reduction. It was therefore considered that the retention of Cu-PTSM in the brain is closely related to mitochondrial reduction, most probably involving the mitochondrial electron transport system.

In vitro investigations on cellular damage induced by high energy shock waves.

Single-cell suspensions of the prostate carcinoma cell line PCA were exposed to electromagnetically generated ultrasound shock waves (source and focusing lens identical to those used in the commercially available lithotripor Lithostar Plus). Cell loss of up to 40% occurred in sample tubes containing air. To expose multicellular tumor spheroids and cells growing on a microcarrier, an experimental setup was developed that prevented motion of the specimen. Intracellular damage of intact spheroids was analyzed by laser scanning microscopy following specific fluorescence staining. Different sensitivities of individual cell components with respect to the applied energy density of the pulses were found, namely defects on cell membranes (0.12 mJ/mm2), vimentin (0.21 mJ/mm2), mitochondria (0.33 mJ/mm2) and nuclear membranes (0.5 mJ/mm2). Loss of cells growing on a microcarrier was found after application of 200 pulses with 0.21 mJ/mm2.

[Ultrastructural study of adenolymphoma].

Ultrastructure of 10 adenolymphomas of parotid gland were studied. The neoplastic epithelium consists of "oncocytes" which are arranged in pseudostratified pattern. Their cytoplasm is almost totally packed with a large numbers of hypertrophic mitochondria showing a variety of peculiar forms. The amount and alteration of the mitochondria gradually increase in the order of pyramidal cell, common columnar cell and degenerated columnar cell. This might be a process of transposition representing degeneration due to metabolic disorder in the tumor. The obvious alterations of mitochondria are found in the cases with not only "hot" nodule but also "cold" nodule shown in the studies of 99mTc scintigraphy. There seems no relationship between "hot" nodule and alteration of mitochondria.