Zhenlei Xia

Tricyclic antidepressants inhibit IL-6, IL-1β and TNF-α release in human blood monocytes and IL-2 and interferon-γ in T cells

Tricyclic antidepressants (TCAs) are widely used in treating depressive disorders. It has been demonstrated that, for instance, IL-1 beta and IL-6 inhibit the HPA axis, which plays a role in the development of depressions. Therefore. we were interested in investigating how TCAs influence cytokine release by T lymphocytes and monocytes respectively. Cells were incubated with either 5 microM clomipramine, 15 microM imipramine or 20 microM citalopram. IL-2 release was suppressed to 60% of the control values by clomipramine and imipramine (p = 0.001; p = 0.000), but citalopram was found to cause a much weaker inhibition (only 18%) (p = 0.16). INF-gamma release was affected to a lower degree than IL-2 release, and imipramine (34%) (p = 0.054) was more potent than clomipramine (24%) (p = 0.16) and citalopram (12%) (p = 0.059) in this case. Monocytes incubated with TCA for 4 h exhibited only limited inhibition of IL-1 beta and IL-6 release, i.e., 6-25% for all three compounds. The corresponding value for TNF-alpha release was 20-45% inhibition, with citalopram being the weakest inhibitor. After 10 h of monocytes to LPS exposure, all three compounds exerted a strong inhibition of IL-1 beta and TNF-alpha release, i.e., 60-70% with p-values below 0.012 for all of them. However the inhibition of IL-6 release was less than 35%. Citalopram was equality as potent as imipramine and clomipramine in inhibiting IL-6 release after long-term exposure of monocytes to LPS. All three TCAs elevated intracellular cAMP concentrations significantly in T lymphocytes and monocytes (p < 0.001).

Changes in the generation of reactive oxygen species and in mitochondrial membrane potential during apoptosis induced by the antidepressants imipramine, clomipramine, and citalopram and the effects on these changes by Bcl-2 and Bcl-XL

In order to investigate the molecular mechanism of the antineoplastic effects exerted by the antidepressive agents imipramine, clomipramine, and citalopram, we examined the effects of these compounds on cell viability, generation of reactive oxygen species (ROS), and mitochondrial membrane potential (DeltaPsi(m)) in human acute myeloid leukemia HL-60 cells. Our results indicate that exposure to these compounds causes a loss in cell viability by activating the apoptotic process, as identified by electron microscopy, DNA gel electrophoresis, and flow cytometry. The increased generation of ROS induced by these drugs was a relatively early event and preceded the loss of DeltaPsi(m). Overexpression of the antiapoptotic protein Bcl-2 or Bcl-X(L) prevents antidepressant-induced apoptosis, as well as loss of DeltaPsi(m), but does not affect the generation of ROS.

The antidepressants imipramine, clomipramine, and citalopram induce apoptosis in human acute myeloid leukemia HL-60 cells via caspase-3 activation

Some widely used antidepressants such as imipramine, clomipramine, and citalopram have been found to possess antineoplastic effects. In the present study, these compounds were found to induce apoptotic cell death in human acute myeloid leukemia HL‐60 cells. Apoptosis induced by the antidepressants was identified by electron microscopy and conventional agarose gel electrophoresis and was quantitated by propodium iodide staining and the terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling (TUNEL) via flow cytometry. Treatment with apoptosis‐inducing concentrations of the antidepressants (80 μM imipramine, 35 μM clomipramine, or 220 μM citalopram) caused induction of caspase‐3/caspase‐3‐like activity, which was monitored by the cleavage of poly(ADP‐ribose) polymerase (PARP), the loss of the 32 kD caspase‐3 (CPP32) precursor, and the cleavage of the fluorescent CPP32‐like substrate PhiPhiLux. Pretreatment with a potent caspase inhibitor benzyloxycarbonyl‐Val‐Ala‐Asp‐fluoromethyl‐ketone (zVAD‐fmk) inhibited antidepressant‐induced CPP32/CPP32‐like activity and apoptosis. Furthermore, activation of caspase induced by the antidepressants was preceded by the hypergeneration of intracellular reactive oxygen species (ROS). These results suggested that the antidepressants may induce apoptosis via a caspase‐3‐dependent pathway, and induction of apoptosis by the antidepressants may provide a clue for the mechanism of their antineoplastic effects.

Antidepressant-induced lipidosis with special reference to tricyclic compounds

Cationic amphiphilic drugs, in general, induce phospholipid disturbances. Tricyclic, as well as other antidepressants belong to this group. In experimental animals, antidepressants induce lipid storage disorders in cells of most organs, a so-called generalized phospholipidosis. This disorder is conveniently detected by electron microscopic examination revealing myelin figures. Myelin figures or myeloid bodies are subcellular organelles containing unicentric lamellar layers. The lipidotic induction potency during in vivo is related to the apolarity of the compound. Metabolism of phospholipids takes place within the cell continuously. Several underlying mechanisms may be responsible for the induction of the phospholipid disturbance. For instance, it has been suggested that the compounds bind to phospholipids and such binding may alter the phospholipids suitability as a substrate for phospholipases. Free TCA or metabolites thereof may also inhibit phospholipases directly, as has been demonstrated for sphingomyelinase in glioma and neuroblastoma cells. Both these mechanisms might result in phospholipidosis. Interaction between drug and phospholipid bilayer has been investigated by nuclear magnetic resonance technique. There seems to be large differences in the sensitivities amongst different organs. Steroid-producing cells of the adrenal cortex, testis and ovaries are in particular susceptible to drug-induced lipidosis. The so-called foam cells are lung macrophages located in the interstitium which become densely packed with myelin figures during TCA exposure. It requires about 3-6 weeks of treatment to develop this converted cell. In cell cultures however, phospholipidosis is demonstrated already after 24 h only. It appears that the cells that undergo TCA-induced lipidosis may recover after withdrawal of the drug. The time required to achieve complete recovery ranges from 3-4 weeks to several months, depending on the organ affected. Little is known about the functional significance of lipidosis. Even if TCA and other antidepressants show other effects, it has not been possible to exclusively relate it to phospholipidosis. However, few attempts have been made to correlate the physiological effects of TCAs in experimental animals to the morphological changes associated with phospholipidosis. There is an increasing evidence however, that cationic amphiphilic drugs may have effects on immune function, signal transduction and receptor-mediated events, effects that to some extent might be related to disturbances in phospholipid metabolism.

The atrophy and changes in the cellular compositions of the thymus and spleen observed in mice subjected to short-term exposure to perfluorooctanesulfonate are high-dose phenomena mediated in part by peroxisome proliferator-activated receptor-alpha (PPARα)

We have previously shown that short-term, high-dose exposure of mice to the environmentally persistent perfluorooctanoate (PFOA) results in thymic and splenic atrophy and the attenuation of specific humoral immune responses. Here we characterize the effects of a 10-day treatment with different dietary doses (1-0.001%, w/w) of perfluorooctanesulfonate (PFOS), a similar fluorochemical, on the immune system of male C57BL/6 mice. At doses greater than 0.02%, PFOS induced clinical signs of toxicity in the animals, whereas at the concentration of 0.02%, this compound caused weight loss, hepatomegaly and atrophy of the thymus, spleen and adipose tissue without toxicity. With this latter dose, histopathological and flow-cytometric analysis revealed that (i) the thymic cortex was virtually depleted of cells; (ii) the total numbers of thymocytes and splenocytes were reduced by 84 and 43%, respectively; (iii) although all populations of thymocytes and splenocytes were smaller, the thymic CD4(+)CD8(+) cells and the splenic B-lymphocytes were most decreased. These alterations resembled those evoked by analogous exposure to PFOA, but were less pronounced. At lower doses (less than 0.02%), PFOS induced hepatomegaly without affecting the thymus or spleen. Finally, comparison of male wild-type 129/Sv mice and the corresponding knock-outs lacking peroxisome proliferator-activated receptor-alpha (PPARalpha) indicated that these effects of PFOS are not strain-dependent. More importantly, hepatomegaly is independent of PPARalpha, the thymic changes are partially dependent on this receptor, and splenic responses are largely eliminated in its absence. Thus, immunomodulation caused by PFOS is a high-dose phenomenon partially dependent on PPARalpha.

Modulation of Apoptosis Induced by Tricyclic Antidepressants in Human Peripheral Lymphocytes

We have recently reported that the tricyclic antidepressants (TCAs) imipramine, clomipramine, and citalopram induce apoptosis in human peripheral lymphocytes. This system is well suited for studies on the pathophysiology/physiology of apoptosis regulation. Apoptosis was determined using both DNA gel electrophoresis and flow cytometric analysis. TCA‐induced apoptosis in lymphocytes was monitored in the presence of the protein synthesis inhibitor cycloheximide (CHX), the RNA synthesis inhibitor actinomycin D (Act D), the antioxidant reduced glutathione (GSH), the nuclease inhibitor aurintricarboxylic acid (ATA), the cytokine interlukin‐2 (IL‐2), and the immunostimulator linomide. CHX and Act D failed to prevent and actually enhanced TCA‐induced apoptosis in lymphocytes, indicating that protein and RNA syntheses are not required for this process. Exogenous IL‐2, GSH, and ATA protected the lymphocytes from apoptosis induced by TCAs in a dose‐dependent manner, whereas linomide had no effect on TCA‐induced apoptosis under our in vitro conditions. Our data demonstrate that TCA‐induced apoptosis in human lymphocytes shares many common features with other stimuli‐induced apoptotic processes.

Reduced cell survival and morphological alterations induced by three tricyclic antidepressants in human peripheral monocytes and lymphocytes and in cell lines derived from these cell types

Tricyclic antidepressant drugs are widely used for the treatment of manic-depressive disorders. As such compounds have been reported to give rise to myelin figures in lymphocytes in experimental animals, the effects of clomipramine (2.5-50 muM), imipramine (2.5-100 muM) and citalopram (5-50 muM) on human peripheral lymphocytes, granulocytes and monocytes in culture were investigated. No cytoplasmic alterations were detected in T or B lymphocytes, but large myelin figures could be seen by fluorescence and electron microscopy in monocytes. Optimal concentrations for the formation of these structures were 20 muM for clomipramine and 40 muM for imipramine. A strongly dose-dependent inhibition of the growth of Molt-4 and U937 cells was also observed with clomipramine, which was 2.5-fold as potent as imipramine in this respect. Treated U937, but not Molt-4, cells showed an increased content of fluorescent granules compared with untreated cells. Furthermore, these tricyclic antidepressants appeared to induce apoptosis in lymphocytes, as judged from the disorganization of the nucleus and the appearance of a typical DNA ladder pattern in treated cells.

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.