Tricyclic antidepressant-induced lipidosis in human peripheral monocytes in vitro, as well as in a monocyte-derived cell line, as monitored by spectrofluorimetry and flow cytometry after staining with Nile red

Human mono- and lymphocytes from peripheral blood and the monoblastoid cell line U-937 were used in this in vitro study of drug-induced lipidosis. Mono- and lymphocytes were exposed for 4 days to three different tricyclic antidepressants (TCAs), imipramine (25 microM), clomipramine (10 microM) and citalopram (80 microM). The lipophilic fluorophore Nile red, which stains intracellular lipid structures selectively, was used as a lipid probe. Fluorescence microscopy, spectrofluorimetry and flow cytometry were used to detect cellular lipidosis, as verified by electron microscopy. Our results demonstrate that imipramine, clomipramine and citalopram induce lipidosis in monocytes and U-937 cells, but not in lymphocytes. An accurate quantitation of induced intracellular lipidosis can be achieved by spectrofluorimetric and flow cytometric analysis.     

HMG-CoA reductase inhibitors induce apoptosis in mouse proximal tubular cells in primary culture

Renal cyst formation in polycystic diseases or after nephron reduction is attributed to enhanced tubular cell proliferation with unbalanced cell death. The induction of tubular cell death could be effective to reduce renal cyst formation. In this study, we examined the effects of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors on apoptosis in mouse proximal tubular (MPT) cells in primary culture. After treatment with HMG-CoA reductase inhibitors, the extracted DNA was analyzed by gel-electrophoresis under ultraviolet light. Apoptosis was evaluated quantitatively by estimating the ratio of fragmented DNA over intact DNA. For morphologic studies, cells were stained with Hoechst 33,258. DNA ladder pattern of 200 kDa typical of apoptosis and significant increase in DNA fragmentation were seen after 24 hours of treatment with lovastatin, a HMG-CoA reductase inhibitor. Staining with the Hoechst dye revealed cleavage of nucleus into pieces under the same condition. Geranylgeranylpyrophosphate (20 microM) and mevalonate (500 microM) completely reversed the effect of lovastatin, while farnesylpyrophosphate (20 microM) partially reversed it. Other products of HMG-CoA pathway such as cholesterol, ubiquinone, dolichol, and isopentenyladenine had no effect. Perillic acid and alpha-hydoxyfarnesylphosphonic acid, isoprenylation inhibitors, induced apoptosis of the cells. A treatment with lovastatin caused actin filament disruption. Cytochalasin D, an inhibitor of actin polymerization, induced apoptosis. Interleukin-1 beta-converting enzyme inhibitor II, a protease inhibitor, had no effect on the apoptosis induced by either HRI or cytochalasin D. The present study suggests that in mouse proximal tubules, HMG-CoA reductase inhibitors induce apoptosis via inhibition of isoprenoid production, and disruption of actin filaments may play a role in the apoptosis induction.     

Endothelin and the airway mucosa

Antimony compounds are widely used in various manufacturing and semiconducting industries. Previously, it has been shown that antimony trichloride (SbCl3) elevates sister chromatid exchange (SCE) rates in V79 cells after a 28-h incubation. However, only limited data on its genotoxic effects are available so far. The present results demonstrate that a 4-h exposure to > 50 microM SbCl3 could induce micronuclei (MN) formation in cultured Chinese hamster ovary (CHO-K1) cells, human bronchial epithelial (BES-6) cells and human fibroblasts (HF). The order of sensitivity to SbCl3 determined by Sulforhodamine B (SRB)-staining survival assay is HF > BES-6 cells > CHO-K1 cells, with LD50 values in these cells being approximately 40, 80 and 180 microM, respectively. Apoptosis and DNA fragmentation was not found in cells immediately following 4-h SbCl3 treatment. However, DNA fragmentation was detected in CHO-K1 cells after 4-h SbCl3 treatment and a 16 h or more post incubation in fresh medium by 1.5% agarose gel electrophoresis. The delayed apoptosis was also observed under microscopic examination in HF, BES-6 and CHO-K1 cells after similar treatment protocol. In addition, an increase in calcium accumulation appeared in CHO-K1 cells and HF immediately after a 4-h SbCl3 treatment, or after a 24-h post incubation in fresh medium. The present results provide important genotoxic and cytotoxic information characterizing the cellular changes induced by short-term SbCl3 exposure in rodent and human cells.     

Peroxynitrite-induced apoptosis in epithelial (T84) and macrophage (RAW 264.7) cell lines: effect of legume-derived polyphenols (phytolens)

Peroxynitrite (ONOO-) has been proposed as a mediator of gut inflammation and as an inducer of cell death by apoptosis. Phytolens (PHY), a water-soluble extract of polyphenolic antioxidants from nonsoy legumes (Biotics Research Corp, patent pending), was evaluated as a cytoprotective agent in human colonic (T84) and murine macrophage (RAW 264.7) cell lines. In the antioxidant testing, PHY showed a significant free radical scavenging ability against 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and superoxide (O2.) radicals with an IC50 of 4.44 and 5.87 microg/ml against DPPH and O2., respectively. Apoptosis (DNA fragmentation) was measured by an ELISA technique. Cells were exposed to oxidative stress by treating them with peroxynitrite (100-300 microM) for 4 h in the presence and absence of PHY. Peroxynitrite elicited a dose-dependent increase in DNA fragmentation in both cell lines compared to the control group receiving decomposed ONOO-. PHY (10, 30, or 50 microg/ml) significantly attenuated the degree of apoptosis in T84 cells induced by ONOO- (P < 0.05). PHY (10-100 microg/ml) did not directly affect T84 cell viability or induce apoptosis after 4 h or overnight exposure. RAW 264.7 cells exposed to PHY alone (>30 microg/ml) for 4 h displayed decreased cell viability (P < 0.05) and increased apoptosis (P < 0.05). Phytolens may have beneficial effects on inflammation by attenuating peroxynitrite-induced apoptosis. The sparing of epithelial cells while compromising the viability of macrophages suggests that PHY may be beneficial in autoimmune disorders.     

Dioxin perturbs, in a dose- and time-dependent fashion, steroid secretion, and induces apoptosis of human luteinized granulosa cells

Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) is the most toxic congener of a large class of environmental pollutants. Several studies have shown that TCDD exposure reduced fecundity and ovulatory rate in rats and increased the incidence of endometriosis in monkeys. Recent work suggests that TCDD's endocrine-disrupting effects are, at least in part, caused by a direct action at the ovary. Although the factors involved in TCDD-induced toxicity are still under investigation, several studies have shown that TCDD induces programmed cell death, or apoptosis, in various tissues and may act in a similar fashion in the ovary. In the present study, we set out to evaluate the in vitro effects of TCDD on steroid secretion, specifically estradiol-17beta (E2) and progesterone, by human luteinized granulosa cells (LGC), and to further determine whether TCDD is capable of inducing apoptosis in this cell type. Human LGC were obtained from women participating in an in vitro fertilization program. Medium, with or without three different concentrations of TCDD and substrates [androstenedione (A4) or pregnenolone], was added to each culture. The media were collected at 4, 8, 12, 24, 36, and 48 h and were assayed by RIA. At 24 and 48 h, the LGC were fixed for assessment of DNA fragmentation via an in situ immunofluorescence technique. Transmission electron microscopy was also performed on LGC after 24 and 48 h with TCDD. TCDD, at all concentrations tested (3.1 pM, 3.1 nM, and 3.1 microM), significantly reduced E2 accumulation in the media at 8, 12, and 24 h, compared with controls. At 36 and 48 h, TCDD treatment (at 3.1 microM) caused a significant increase in E2, compared with controls. The effect of TCDD on E2 was abolished with the addition of A4. TCDD treatment did not alter progesterone accumulation. Apoptosis increased at 24 h with 3.1 microM TCDD, with no apparent effect at 3.1 nM. By 48 h, however, TCDD increased apoptosis in a dose-dependent manner. Transmission electron microscopy showed ultrastructural differences in LGC with 3.1 microM TCDD at 24 and 48 h. Collectively, the results of the present study suggest that TCDD perturbs E2 secretion by depletion of A4 precursor and increases apoptotic cell death of human LGC in a dose- and time-dependent fashion.     

Levels of monoamine and amino acid neurotransmitters in the developing male mouse hypothalamus and in histotypic hypothalamic cultures

PURPOSE: Cytosine arabinoside induces apoptosis and this cell death process is influenced by protein kinase C signaling events in leukemic cells. We present findings that extend these observations to include another deoxycytidine analog, gemcitabine, which is more potent in solid tumors. METHODS AND RESULTS: Gemcitabine induced programmed cell death in BG-1 human ovarian cancer cells based on biochemical and morphologic analyses. The DNA was fragmented in BG-1 cells exposed to gemcitabine (0.5 microM, 1.0 microM and 10 microM) for 8 h, but gemcitabine treatment did not induce internucleosomal DNA degradation. Scanning and transmission electron microscopy of BG-1 cells showed morphologic changes associated with apoptosis in response to gemcitabine: membrane blebbing, the formation of apoptotic bodies and chromatin condensation. Thus, BG-1 cells undergo programmed cell death in response to gemcitabine treatment without internucleosomal DNA fragmentation. Furthermore, gemcitabine (10 microM) activated protein kinase C in BG-1 cells and the phosphorylation of the endogenous protein kinase C substrate, myristoylated alanine-rich C kinase substrate, was increased following exposure of BG-1 cells to gemcitabine for up to 6 h. Clonogenicity studies with gemcitabine in combination with various protein kinase C-modulating agents demonstrated that gemcitabine cytotoxicity was influenced by protein kinase C signaling events in BG-1 cells. Short-term (1 h) exposure to TPA (1 or 10 nM) followed by gemcitabine (0.5 microM for 4 h) did not alter the response to gemcitabine. However, a 24-h exposure to TPA followed by gemcitabine resulted in synergistic cytotoxicity, while coincubation of TPA with a PKC inhibitor (e.g. bisindolylmaleimide or calphostin-C) in this regimen abrogated the synergistic response. CONCLUSIONS: Based on our findings, it is plausible that gemcitabine therapy could be improved by modulating PKC signaling events linked to drug-induced apoptosis/cytotoxicity.     

Dysregulation of bcl-2, c-myc, and Fas expression during tricyclic antidepressant-induced apoptosis in human peripheral lymphocytes

Tricyclic antidepressants (TCAs) have been shown to induce apoptosis in human lymphocytes. In the present report, we investigated in parallel the regulation of the three oncogenes bcl-2, c-myc, and Fas. A reduction in c-myc and bcl-2 levels of 35-40% and 22-27%, respectively, was observed. On the other hand, Fas expression on the outer surface of the plasma membrane was increased up to 31%. In conclusion, bcl-2, c-myc, and Fas are undergoing dysregulation due to TCA-induced apoptosis.     

Tissue equivalent vessel phantoms for intravascular ultrasound

The presence of the non-selective protein kinase C (PKC) inhibitors, staurosporine (100 nM) and polymyxin B (100 microM) in cultured human RPE cells for more than 24 h triggers apoptotic death. Apoptosis is characterized by a diminishing number of cells, a labelling of nuclei by the TUNEL method and by observable morphological changes. An inhibitor of PKC and cyclic nucleotide-dependent protein kinases, 1-(5-isoquinolinesulphonyl)-2-methyl piperazine (H-7; 100 microM), was without effect, as was the specific PKC inhibitor, calphostin C (100 nM). The PKC-activating phorbol esters, phorbol-12-myristate-13-acetate (PMA; 1 microM) and phorbol-12,13-dibutyrate (PDB; 1 microM) and the non-tumour-promoting phorbol ester, 4 alpha-PMA (1 microM) were without effect, as was the diacyl glycerol analogue, 1,2-dioctanoyl-snglycerol (DOG; 10 microM). The PKC activators did not attenuate the apoptosis induced by staurosporine or polymyxin B. Furthermore, deprivation of glucose and oxygen (simulated ischemia) for 72 h induced apoptosis: this could be prevented by inclusion of 10% (v/v) foetal bovine serum (FBS) but not by a variety of PKC activators. Six PKC isoenzymes were shown to be present in RPE cells (alpha, beta 1, beta 2, delta, epsilon, E) and only the calcium-dependent cPKC levels changed after treatment with staurosporine or simulated ischaemia. Since only the less selective inhibitors of PKC induced apoptosis, it is suggested that PKC is not involved directly in the induction process of apoptosis in RPE cells. It is possible that the staurosporine and polymyxin B-induced effects of apoptosis in RPE cells are triggered by an unknown kinase-dependent pathway, but whether the 'ischaemia'-induced death is related to this same process remains to be elucidated.     

Focal adhesion kinase-dependent apoptosis of melanoma induced by tyrosine and phenylalanine deficiency

We found previously that restriction of tyrosine (Tyr) and phenylalanine (Phe) inhibited growth and metastasis of B16BL6 murine melanoma and arrested these cells in the G0-G1 phase of the cell cycle. Here, we report that deprivation of these two amino acids in vitro induces apoptosis in B16BL6 and in human A375 melanoma cells but not in nontransformed, neonatal murine epidermal cells or human infant foreskin fibroblasts. Four days after deprivation of Tyr and Phe in vitro, 37% of B16BL6 and 51% of A375 melanoma cells were undergoing apoptosis. Apoptosis was not associated with elevation in intracellular calcium or alteration in p53 or c-myc protein expression. Expression and Tyr phosphorylation of focal adhesion kinase (FAK) were inhibited in both melanoma cell lines by deprivation of Tyr and Phe but not by deprivation of glutamine or serum. Tyr phosphorylation of FAK in Tyr- and Phe-deprived melanoma cells was enhanced within 30 min of refeeding with complete DMEM. FAK protein expression recovered within 60 min, and cell viability recovered within 24 h. Genistein, a tyrosine kinase inhibitor that specifically inhibits Tyr phosphorylation of FAK, did not induce apoptosis in A375 melanoma cells at a concentration of 50 microM. Genistein prevented the recovery of cell viability upon refeeding with Tyr and Phe to previously deprived A375 melanoma cells. These data collectively indicate that apoptosis induced by Tyr and Phe deprivation is FAK-dependent.     

Effect of trans-retinoic acid and folic acid on apoptosis in human gastric cancer cell lines MKN-45 and MKN-28

Induction of apoptosis has been implicated as an anticarcinogenic mechanism of both folic acid and retinoic acid. The ability of retinoic acid or folic acid to induce gastric cancer cell apoptosis was investigated in the human gastric cancer cell lines MKN-45 and MKN-28, and DNA fragmentation was studied in situ by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling and DNA agarose gel electrophoresis. The rates of apoptosis in both the poorly differentiated MKN-45 and the well differentiated MKN-28 cell line were less than 5% after treatment with either retinoic or folic acid. Apoptosis may be induced by the administration of retinoic acid or folic acid, and the apoptosis indices of MKN-45 and MKN-28 cells were related to the doses of these drugs. The induction of gastric cancer cell apoptosis may play a role in the anticarcinogenic effect of retinoic acid and folic acid, both of which are potential agents for the treatment of human gastric cancer.     

Camptothecin causes cell cycle perturbations within T-lymphoblastoid cells followed by dose dependent induction of apoptosis

We have investigated the effect of the anticancer compound, camptothecin on Jurkat T-cells, a lymphoblastoid leukemic cell-line. Exposure to low concentrations led to rapid cessation of DNA (more than 95%) and RNA (more than 75%) synthesis. Perturbations to the cell cycle were observed following exposure which caused a significant accumulation of cells within G1 (P = 0.03) with a concomitant decrease in G2/M (P = 0.025). Concentrations below 0.1 microM could inhibit DNA synthesis but not induce apoptosis. Induction of apoptosis was dose dependent and could be detected as early as 3 h post exposure. The apoptotic population appeared to be derived from G1 and S-phase cells but not G2/M, coinciding with the cell cycle compartments in which DNA and RNA polymerases function. However, direct inhibition of DNA polymerase alone was not shown to be associated the induction of apoptosis or with a decrease in susceptibility to camptothecin-induced cell death. The effects of camptothecin on Jurkat T-cells and the potential mechanisms involved are discussed in the context of observations made in other transformed cell lines.     

Spontaneous apoptosis in human colon tumor cell lines and the relation of wt p53 to apoptosis

OBJECTIVE: To examine spontaneous apoptosis of cultured human colon tumor cell lines in vitro and to investigate the role of wild type (wt) p53 in regulation of apoptosis induced by DNA-damaging treatment. METHODS: A model system of human tumor progression involving three cell lines was used in this study for examination of apoptosis. They were originally established from human colon villous adenoma, including an early passage of non-tumorigenic cell line, V235E; a late passage of weakly tumorigenic cell line, V235L; and a spontaneous progressing highly tumorigenic cell line. V411. All of them maintain wt p53 expression. For identification of apoptosis, two tests were performed: 1. morphology study using acridine orange (AO)/ethidium bromide (EB) stainning by fluorescence microscopy; 2. DNA electrophoresis on agarose gel. P53 and WAF-1 (a downstream gene of p53) expressions were analysed at mRNA level using Northern blot technique. Apoptotic index of cell lines examined was measured by DNA fluorescence assay. RESULTS: Spontaneous apoptosis was demonstrated in cell lines of all stages of progression by both morphology and DNA agarose gel electrophoresis. Apoptosis was further induced in V411 after treatment of cells with 137Cs gamma-irradiation and accompanied by increases in p53 and WAF-1 expression. In contrast, a mutant p53 bearing human colon cancer cell line, sw480, lacked spontaneous apoptosis, and upon irradiation neither induction of apoptosis nor increase expression of p53 and WAF-1 were seen. CONCLUSIONS: Apoptosis can be maintained in some human tumor cell lines despite transformation and carcinogenesis. Wt p53 and WAF-1 products are two of the potential mediators which effect apoptosis. Additionally, since apoptosis was enhanced by irradiation in V411, but not in sw480, it suggests that wt p53 cancer cells are more sensitive to DNA-damaging treatment than mutant p53 cancer cells. These finding may have implications for cancer therapy.     

Induction of apoptosis by benzene metabolites in HL60 and CD34+ human bone marrow progenitor cells

Two cell types, HL60 human promyelocytic leukemia cells and CD34+ human bone marrow progenitor cells, were used as model systems to explore a possible role for apoptosis in the myelotoxicity of the phenolic metabolites of benzene. HL60 cells were treated with either phenol, catechol, hydroquinone, or 1,2,4-benzenetriol and then stained with Hoechst 33342 and propidium iodide and subjected to fluorescent microscopy. Cells with nuclear condensation and fragmentation were scored as apoptotic, and etoposide (40 microM) was used as a positive control. Catechol, 1,2,4-benzenetriol, and hydroquinone induced marked time- (0-24 hr) and concentration- (25-100 microM) dependent apoptosis, whereas phenol (750 microM) did not. Under these conditions, no significant necrosis was observed. The induction of apoptosis was confirmed by internucleosomal cleavage of DNA, assessed by agarose gel electrophoresis. CD34+ cells treated with etoposide (40 microM) or hydroquinone (50 microM) for 18 hr were stained and subjected to fluorescent microscopy as above. The percentage of cells exhibiting nuclear condensation and/or fragmentation as well as high intensity staining significantly increased in both cases. The induction of apoptosis was confirmed using a terminal deoxynucleotidyl transferase assay. These data show that apoptosis can be induced in both HL60 and CD34+ human bone marrow progenitor cells by benzene metabolites. The ability of phenolic metabolites of benzene to induce apoptosis in human bone marrow progenitor cells may contribute to benzene myelotoxicity.     

Mechanisms involved in sodium arsenite-induced apoptosis of human neutrophils

Apoptosis is a distinct mechanism by which eukaryotic cells die. Factors governing the induction of polymorphonuclear leukocyte (PMN) apoptosis should be important in understanding resolution of acute inflammation. The mechanisms for induction of PMN apoptosis remain uncertain; however, oxidative stress has been suggested. The aims of this study were to determine whether reactive oxygen intermediates play a role in PMN apoptosis and to investigate inhibition of this process by selective use of antioxidants. PMN were isolated from 10 healthy volunteers. PMN (1 x 10(6) PMN/mL) were cultured in 40, 80, and 160 microM of arsenite for 2, 6, 12, 18, and 24 h. Apoptosis was assessed qualitatively by morphology and gel electrophoresis and quantitatively by CD16 receptor expression and propidium iodide DNA staining. There was a significant (P < 0.05) increase in the rate of apoptosis on incubation with arsenite (80 and 160 microM). To investigate the mechanism of this process, intracellular respiratory burst activity was measured following arsenite culture. We found that arsenite-induced PMN apoptosis correlated with an increase in intracellular respiratory burst. To further investigate the role of oxidative injury in inducing apoptosis, the antioxidants catalase, dimethyl sulfoxide (DMSO), glutathione (GSH), N-acetylcysteine (NAC), and taurine were investigated and we demonstrated that GSH, NAC, and taurine were significantly protective against arsenite-induced apoptosis. However, catalase and DMSO failed to induce protection. This study demonstrates that arsenite induces PMN apoptosis through an oxygen-dependent mechanism that can be prevented through selective antioxidants.     

Frequency-shaped amplification changes the neural representation of speech with noise-induced hearing loss

Apoptosis is a mode of active cell death. We have examined whether 2-chloroethylethyl sulfide (CEES), a sulfur vesicating agent, triggers apoptosis as a cytotoxic mechanism. Incubation of thymocytes with CEES, resulted in an induction of apoptotic features of cell death. Treatment of cells with 100 microM CEES for 5 h increased DNA fragmentation to approximately 40% of control. The fragmentation of DNA was visualized by agarose gel electrophoresis. It showed ladder pattern of DNA fragmentation, which indicates internucleosomal cleavage of DNA. Further evidence of apoptosis was observed in morphological changes of nuclei by using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method. The percentage of TUNEL positive cells was dependent upon CEES concentrations. CEES induced the classical morphological features of apoptosis in nucleus. These features were accompanied by condensation of chromatin, which arranged in sharply declined clumps and fragmentation of nucleus. To study requirement for synthesis of new protein in CEES-induced apoptosis, we studied the effect of cycloheximide for apoptotic activity. This protein synthesis inhibitor did not suppress the CEES-induced apoptotic activity. Taken together, these results suggest that CEES-induced apoptosis as a cytotoxicmechanism and this process occurs independent of synthesis of new protein.     

Induction of apoptosis by a short-chain neuropeptide analog in small cell lung cancer

Small cell lung cancer (SCLC) cells express a variety of neuropeptides which act as autocrine growth factors. Although several neuropeptide analogs have been reported to antagonize SCLC proliferation, the development of these compounds has been limited by their low potency and the cytostatic nature of their effects. In the present study we evaluated the cytotoxic activity of four short-chain substance P analogs (NY3460, NY3238[-pHOPA], NY3238[Phe1], NY3238[Lys5]) against a panel of five SCLC cell lines. NY3460 was the most potent compound in all five SCLC cell lines (IC50 = 2.8-3.7 microM) as assessed by a MTT growth inhibitory assay. NY3238[Phe1] was also relatively active in all cell lines (IC50 = 3.5-11.2 microM), while NY3238[Lys5] and NY3238[-pHOPA] were substantially less active. NY3460 was the only agent to induce an increase in the percentage of cells with subdiploid DNA content suggestive of apoptosis by flow cytometric DNA content analysis. The induction of apoptosis was confirmed by fluorescent microscopy in NCI-H69, NCI-H82, NCI-H446, and NCI-H510 cells after exposure to 5.0 microM NY3460 for 48 h. These findings suggest that NY3460 is a relatively potent cytotoxic inhibitor of SCLC growth, and that short-chain neuropeptide analogs deserve further evaluation as anti-SCLC agents.     

Tumor necrosis factor-alpha induces apoptosis of human adipose cells

Tumor necrosis factor-alpha (TNF-alpha) production by adipocytes is elevated in obesity, as shown by increased adipose tissue TNF-alpha mRNA and protein levels and by increased circulating concentrations of the cytokine. Furthermore, TNF-alpha has distinct effects on adipose tissue including induction of insulin resistance, induction of leptin production, stimulation of lipolysis, suppression of lipogenesis, induction of adipocyte dedifferentiation, and impairment of preadipocyte differentiation in vitro. Taken together, these effects all tend to decrease adipocyte volume and number and suggest a role for TNF-alpha in limiting increase in fat mass. The aim of the present study was to determine if TNF-alpha could induce apoptosis in human adipose cells, hence delineating another mechanism by which the cytokine could act to limit the development of, or extent of, obesity. Cultured human preadipocytes and mature adipocytes in explant cultures were exposed in vitro to human TNF-alpha at varying concentrations for up to 24 h. Apoptosis was assessed using morphological (histology, nuclear morphology following acridine orange staining, electron microscopy) and biochemical (demonstration of internucleosomal DNA cleavage by gel electrophoresis and of annexin V staining using immunocytochemistry) criteria. In control cultures, apoptotic indexes were between 0 and 2.3% in all experiments. In the experimental systems, TNF-alpha induced apoptosis in both preadipocytes and adipocytes, with indexes between 5 and 25%. Therefore, TNF-alpha induces apoptosis of human preadipocytes and adipocytes in vitro. In view of the major metabolic role of TNF-alpha in human adipose tissue, and the knowledge that adipose tissue is dynamic (with cell acquisition via preadipocyte replication/differentiation and cell loss via apoptosis), these findings describe a further mechanism whereby adipose tissue mass may be modified by TNF-alpha.