Cytotoxicity and genotoxicity of phenazine in two human cell lines

Phenazine was recently identified as a drinking water disinfection byproduct (DBP), but little is known of its toxic effects. We examined in vitro cytotoxicity and genotoxicity of phenazine (1.9–123 μM) in HepG2 and T24 cell lines. Cytotoxicity was determined by an impedance-based real-time cell analysis instrument. The BrdU (5-bromo-2′-deoxyuridine) proliferation and MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) viability assays were used to examine mechanisms of cytotoxicity. Genotoxicity was determined using the alkaline comet assay. Concentration-dependent cytotoxicity was observed in HepG2 cells, primarily due to an antiproliferative effect (BrdU 24 h IC₅₀: 11 μM; 48 h IC₅₀: 7.8 μM) observed as low as 1.9 μM. T24 cells experienced a minor antiproliferative effect (BrdU 24 h IC₅₀: 47 μM; 48 h IC₅₀: 17 μM). IC₅₀ values for HepG2 proliferation and viability were 54–77% lower compared to T24 cells. In both cell lines, IC₅₀ values for proliferation were 66–90% lower than those for viability. At phenazine concentrations producing equivalent cytotoxicity, HepG2 cells (1.9–30.8 μM) experienced no significant genotoxic effects, while T24 cells (7.7–123 μM) experienced significant genotoxicity at ⩾61.5 μM. While these effects were seen at phenazine concentrations above those found in disinfected water, the persistence of the antiproliferative effect and the differential toxicity in each cell line deserves further study.     

Oxidative stress resulting from exposure of a human salivary gland cells to paraoxon: An in vitro model for organophosphate oral exposure

Organophosphate (OP) compounds are used as insecticides, acaricides, and chemical agents and share a common neurotoxic mechanism of action. The biochemical alterations leading to many of the deleterious effects have been studied in neuronal cell lines, however, non-neuronal toxic effects of OPs are far less well characterized in vitro, and specifically in cell lines representing oral routes of exposure. To address this void, the human salivary gland (HSG) cell line, representing likely interactions in the oral cavity, was exposed to the representative OP paraoxon (PX; O,O-diethyl-p-nitrophenoxy phosphate) over a range of concentrations (0.01–100 μM) and analyzed for cytotoxicity. PX induced cytotoxicity in HSG cells at most of the exposure concentrations as revealed by MTT assay, however, the release of LDH only occurred at the highest concentration of PX tested (100 μM) at 48 h. Slight increases in cellular ATP levels were measured in PX-exposed (10 μM) HSG cells at 24 h. Exposing HSG cells to 10 μM PX also led to an increase in DNA fragmentation prior to loss of cellular membrane integrity implicating reactive oxygen species (ROS) as a trigger of toxicity. The ROS genes gss, gstm2, gstt2 and sod2 were upregulated, and the presence of superoxide following 10 μM PX exposure was determined via dihydroethidium fluorescence studies further implicating PX-induced oxidative stress in HSG cells.     

Carbonyl stress and a combination of astaxanthin/vitamin C induce biochemical changes in human neutrophils

The purpose of the present study was to find out whether co-treatment of human neutrophils with high glucose and methylglyoxal (MGO) can alter the biochemical parameters of human neutrophils. We also examined if astaxanthin associated with vitamin C can improve those biochemical parameters. Neutrophils from healthy subjects were treated with 20 mM of glucose and 30 μM MGO followed or not by the addition of the antioxidants astaxanthin (2 μM) and vitamin C (100 μM). MGO/high glucose treatment reduced the phagocytic capacity and the G6PDH, total/SOD and GR activities. Additionally, there was an increase in the activity of myeloperoxidase (MPO) with consequent increase in the hypochlorous acid production, CAT activity and in the release of IL-6 cytokine without changes in intracellular calcium mobilization. Our study also shows that the association of astaxanthin with vitamin C greatly improved neutrophil phagocytic capacity, decreasing all reactive oxygen species measured, pro-inflammatory IL-1β and TNF-α release, MPO activity and HClO production. The combination of astaxanthin with vitamin C alone has more antioxidant and anti-inflammatory effects than when they were in the presence of MGO/high glucose. Injury to the function of neutrophils due to high glucose and methylglyoxal appears not to involve oxidative stress or calcium release. The association of antioxidants astaxanthin and vitamin C promoted a significant improvement in the function of neutrophils and in the redox status.     

Inhibition of sphingosine kinase-2 ablates androgen resistant prostate cancer proliferation and survival

BackgroundEndogenous sphingolipid signaling has been shown to play an important role in prostate cancer endocrine resistance.MethodsThe novel SphK2 inhibitor, ABC294640, was used to explore SphK signaling in androgen resistant prostate cancer cell death signaling.ResultsIt dose-dependently decreased PC-3 and LNCaP cell viability, IC₅₀ of 28 ± 6.1 μM (p < 0.05) and 25 ± 4.0 μM (p < 0.05), respectively. ABC294640 was more potent in long-term clonogenic survival assays; IC₅₀ of 14 ± 0.4 μM (p < 0.05) in PC-3 cells and 12 ± 0.9 μM (p < 0.05) in LNCaP cells. Intrinsic apoptotic assays failed to demonstrate increased caspase-9 activity. Ki-67 staining demonstrated decreased proliferation by 50 ± 8.4% (p < 0.01) in PC-3 cells.ConclusionsSphK2 inhibition decreases androgen resistant prostate cancer viability, survival, and proliferation independently of the intrinsic apoptotic pathway. Findings are in contrast to recent observations of ABC29460 acting dependently on the intrinsic pathway in other endocrine resistant cancer cell lines.     

Maturin acetate from Psacalium peltatum (Kunth) Cass. (Asteraceae) induces immunostimulatory effects in vitro and in vivo

Maturin acetate (MA) is one of main constituents in Psacalium peltatum. The cytotoxic effects of MA on tumorigenic cells were evaluated using the MTT assay. The in vitro immunostimulatory effects of maturin acetate (MA) were evaluated on the viability of murine splenocytes and macrophages, and human peripheral blood mononuclear cells (PBMC). The effects of MA on the production of nitrous oxide, pinocytosis and lysosomal enzyme activity were assayed in murine macrophages RAW 264.7. The effects of MA on the NK cell activity were also assayed. The in vivo immunostimulatory activities of MA were evaluated on BALB/c mice immunosuppressed with cyclophosphamide (CY). MA lacks cytotoxic activity against human cancer cells (IC₅₀ > 200 μM). In the absence of LPS, MA 10 μM or higher stimulated significantly (P ? 0.05), compared to untreated cells (-LPS), the viability of murine macrophages and splenocytes. In the absence of LPS, MA 10 μM or higher stimulated significantly (P ? 0.05), compared to untreated cells (-LPS), the lysosomal enzyme activity and pinocytosis. In immunosuppressed mice, MA increases significantly (P ? 0.05), compared to CY-treated mice, the production of IL-2 and IL-15 and IFN-γ. In conclusion, MA exerts immunostimulatory activities in vitro and in vivo.     

Interactions between dietary flavonoids apigenin or luteolin and chemotherapeutic drugs to potentiate anti-proliferative effect on human pancreatic cancer cells,in vitro

The objectives were to assess the potential of dietary flavonoids apigenin (Api) and luteolin (Lut) to enhance the anti-proliferative effects of chemotherapeutic drugs on BxPC-3 human pancreatic cancer cells and to investigate the potential molecular mechanism of action. Simultaneous treatment or pretreatment (0, 6, 24 and 42 h) of flavonoids and chemotherapeutic drugs at various concentrations (0–50 μM) were assessed using the MTS cell proliferation assay. Simultaneous treatment with either flavonoid (13, 25 or 50 μM) and chemotherapeutic drugs 5-fluorouracil (5-FU, 50 μM) or gemcitabine (Gem, 10 μM) for 60 h resulted in mostly less-than-additive effects (p < 0.05). Pretreatment for 24 h with 13 μM of either Api or Lut, followed by Gem for 36 h was optimal to inhibit cell proliferation. Pretreatment of cells with 11–19 μM of either flavonoid for 24 h resulted in 59–73% growth inhibition when followed by Gem (10 μM, 36 h). Lut (15 μM, 24 h) pretreatment followed by Gem (10 μM, 36 h), significantly decreased protein expression of nuclear GSK-3β and NF-κB p65 and increased pro-apoptotic cytosolic cytochrome c. Pretreatment of BxPC-3 human pancreatic cancer cells with low concentrations of Api or Lut effectively aid in the anti-proliferative activity of chemotherapeutic drugs.     

In vitro impact of bisphenols BPA,BPF, BPAF and 17β-estradiol (E2) on human monocyte-derived dendritic cell generation,maturation and function

Bisphenols (BPs) are widely spread pollutants that act as estrogen-like endocrine disruptors and are potentially affecting human health on a long run. We explored the effects of BPA, BPF and BPAF, on in vitro differentiation and maturation of MDDCs. Monocytes were treated with 17β-estradiol (E2) and each BP at the beginning of their differentiation into iMDDCs. We found that 10 and 50 μM of BPA and BPF, 10 and 30 μM of BPAF and 10 and 50 nM of E2 did not affect cell viability. However, 50 μM of BPA and BPF, as well as 10 and 30 μM of BPAF, significantly decreased the endocytotic capacity of iMDDCs. Both, BPA (50 μM) and BPAF (30 μM) decreased the expression of CD1a and increased the amount of DC-SIGN molecules on iMDDCs. The E2 pre-treatment moderately decreased expression of CD80, CD86 and CD83 co-stimulatory molecules while increasing the numbers of HLA-DR on mMDDCs. Only BPAF significantly influenced the expression of CD80 and CD86 (both decreased), as well as CD83 and HLA-DR molecules (both increased) on mMDDCs. In addition, BPAF modulated DC maturation signaling pathways by lowering the phosphorylation of p65 NF-κB (nuclear factor-kappaB) and ERK (extracellular signal regulated kinase) 1/2 proteins. Consequently, the in vitro proliferation of allogeneic T cells, stimulated with differently pre-treated iMDDCs and mMDDCs, was significantly reduced only in case of BPAF.     

Developmental cardiotoxicity effects of four commonly used antiepileptic drugs in embryonic chick heart micromass culture and embryonic stem cell culture systems

Antiepileptic drugs (AEDs) are commonly used drugs in pregnant women with epilepsy. Prenatal exposure to AEDs increases the risk of major or minor congenital malformation during embryonic development. The precise mode of action and intracellular mechanisms of these AEDs during embryonic development remains unclear. To determine relative teratogenic risk of AEDs, four AED drugs including valproic acid (VPA), phenytoin (PHT), phenobarbital (PB), and trimethadione (TMD) were tested using two in vitro systems (the embryonic chick heart micromass (MM) culture and the in vitro differentiating mouse embryonic stem cells into cardiomyocytes culture systems). Cardiomyocyte cultures (the heart MM and ES cell-derived cardiomyocytes) were treated with or without different concentrations of PHT, PB, TMD (10–100 μM), and VPA (100–2000 μM). 5-Fluorouracil (5-FU) (1–10 μM) and l-ascorbic acid (10–1000 μM) were used as positive and negative controls. It was found that these four commonly used AEDs and 5-FU tested have the potential to inhibit embryonic heart cell differentiation (p < 0.05) without inducing any cytotoxicity. VPA at higher concentrations (⩾800 μM), and 5-FU at all doses proved to be cytotoxic in the differentiating ES cell culture system. The results demonstrated in this study suggest that the use of these four commonly prescribed AEDs during pregnancy may have an effect on embryonic heart development, and may be associated with congenital cardiovascular defects.     

Effect of vitamin E and C supplementation on oxidative damage and total antioxidant capacity in lead-exposed workers

The molecular response of the antioxidant system and the effects of antioxidant supplementation against oxidative insult in lead-exposed workers has not been sufficiently studied. In this work, antioxidants (vitamin E 400 IU + vitamin C 1 g/daily) were supplemented for one year to 15 workers exposed to lead (73 μg of lead/dl of blood) and the results were compared with those on 19 non-lead exposed workers (6.7 μg of lead/dl). Lead intoxication was accompanied by a high oxidative damage and an increment in the erythrocyte antioxidant response due to increased activity of catalase and superoxide dismutase. Antioxidant supplementations decreased significantly the oxidative damage as well as the total antioxidant capacity induced by lead intoxication with reduction of the antioxidant enzyme activities. We conclude that antioxidant supplementation is effective in reducing oxidative damage and induces modifications in the physiopathological status of the antioxidant response in lead-exposed workers.     

Zinc enhances CDKN2A,pRb1 expression and regulates functional apoptosis via upregulation of p53 and p21 expression in human breast cancer MCF-7 cell

Zinc (Zn) is an essential trace elements, its deficiency is associated with increased incidence of human breast cancer. We aimed to study the effect of Zn on human breast cancer MCF-7 cells cultured in Zn depleted and Zn adequate medium. We found increased cancer cell growth in zinc depleted condition, further Zn supplementation inhibits the viability of breast cancer MCF-7 cell cultured in Zn deficient condition and the IC_25, IC₅₀ value for Zn is 6.2 μM, 15 μM, respectively after 48 h. Zn markedly induced apoptosis through the characteristic apoptotic morphological changes and DNA fragmentation after 48 h. In addition, Zn deficient cells significantly triggered intracellular ROS level and develop oxidative stress induced DNA damage; it was confirmed by elevated expression of CYP1A, GPX, GSK3β and TNF-α gene. Zinc depleted MCF-7 cells expressed significantly (p ≤ 0.001) decreased levels of CDKN2A, pRb1, p53 and increased the level of mdm2 expression. Zn supplementation (IC₅₀ = 15 μM), increased significantly CDKN2A, pRB1 & p53 and markedly reduced mdm2 expression; also protein expression levels of CDKN2A and pRb1 was significantly increased. In addition, intrinsic apoptotic pathway related genes such as Bax, caspase-3, 8, 9 & p21 expression was enhanced and finally induced cell apoptosis. In conclusion, physiological level of zinc is important to prevent DNA damage and MCF-7 cell proliferation via regulation of tumor suppressor gene.     

Effects of endocrine disrupting chemicals on in vitro global DNA methylation and adipocyte differentiation

Recent studies suggest that endocrine disrupting chemicals (EDCs) may form a risk factor for obesity by altering energy metabolism through epigenetic gene regulation. The goal of this study is to investigate the effects of a range of EDCs with putative obesogenic properties on global DNA methylation and adipocyte differentiation in vitro. Murine N2A and human SK-N-AS neuroblastoma cells and murine preadipocyte fibroblasts (3T3-L1) were exposed to tributyltin (TBT), diethylstilbestrol (DES), bisphenol A (BPA), 2,3,7,8-tetrachlorodibenzo-[p]-dioxin (TCDD), 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB-153), hexachlorobenzene (HCB), hexabromocyclododecane (HBCD), 2,2′,4,4′-tetrabrominated diphenyl ether (BDE-47) , perfluorinated octyl acid (PFOA) and perfluorinated octyl sulfonate (PFOS). A modest decrease in global DNA methylation was observed in N2A cells exposed to 10 μM DES, BPA, TCDD, BDE-47, PCB-153 and 1 μM HCB, but no changes were found in the human SK-N-AS cells. We reveal for the first time that BDE-47 increases adipocyte differentiation in a dose-dependent manner (2.5–25 μM). Adipocyte differentiation was also enhanced by TBT (?10 nM) and BPA (>10 μM) and inhibited by TCDD (?0.1 nM). The other chemicals showed either modest or no effects on adipocyte differentiation at the concentrations tested (PFOA, PFOS and HBCD at 10 μM; PCB-153, 3.4 μM and HCB, 1 μM). This study demonstrates that selected EDCs can induce functional changes in murine adipocyte differentiation in vitro which are accompanied by decreased global DNA methylation.     

Teratogenic effects of diabetic conditions in chick heart in ovo and in micromass culture may be prevented by addition of vitamin C and folic acid

Maternal diseases like diabetes mellitus may cause developmental defects. Supplementation with folic acid and vitamin C during the periconceptional period has been shown to prevent some neural tube and congenital heart defects. Hearts were dissected from 5 days-old White Leghorn chick embryos, the cells isolated and cultured in micromass under diabetic conditions, with and without folic acid and vitamin C. Contractile activity, cell viability (resazurin reduction) and protein assays were performed. Results indicated diabetic conditions reduced contractile activity and cell viability, whilst vitamin C (100 μM) and folic acid (1 mM) administered concurrently significantly improved them to values comparable with the control. Day 3 chick embryos in ovo were injected with glucose + hydroxybutyrate or a combination of these and vitamins. Diabetic conditions caused gross and histological malformations, but these effects were abrogated by vitamin supplement. Teratogenic effects on heart development could possibly be prevented by vitamin supplementation during pregnancy.     

Evaluation of a predictive in vitro Leydig cell assay for anti-androgenicity of phthalate esters in the rat

An in vitro assay using the rat Leydig cell line R2C was evaluated for its ability to quantitatively predict inhibition of testosterone synthesis. Results obtained for endocrine active phthalates (MEHP, MBP), and inactive phthalates (MMP and MEP) were highly consistent with in vivo results based on tissue and media concentrations. Statistically significant inhibition of testosterone synthesis (p < 0.05, 1-way ANOVA) was observed at 1 μM MBP and 3 μM MEHP, while MEP and MMP did not affect inhibition of testosterone synthesis until much higher concentrations (?100 μM). Concentrations causing 50% inhibition of testosterone synthesis for MBP and MEHP (3 and 6 μM respectively), were similar to in vivo values (3 and 7 μM). The R2C assay was used to determine the relative potency of 14 structurally diverse monoesters and oxidative metabolites of MEHP. Monoesters with alkyl chains 4–5 carbons in length had the highest potency for testosterone inhibition, while 0–2 carbon alkyl chains were least potent. Phase I metabolism did not completely inactivate MEHP, underscoring the need for metabolism data when interpreting in vitro pharmacodynamic data. This steroid inhibition assay provides a predictive in vitro alternative to expensive and timeconsuming developmental rat studies for phthalate-induced antiandrogenicity.     

The in vitro effects of selenomethionine on methylmercury-induced neurotoxicity

Selenium (Se) has been reported to reduce the severity of MeHg-induced neurological deficits. Therefore, we investigated whether 24 h. preincubation or 50 min. coincubation with selenomethionine (SeMet) was effective in reducing methylmercury (MeHg)-induced cytotoxicity in C6-glioma and B35-neuronal cell lines. As indicators of cytotoxicity, reduced glutathione (GSH), reactive oxygen species (ROS) and mitochondrial activity (MTT) was assessed. Measurement of GSH with the fluorescent indicator MCB-monochlorobimane indicated that in SeMet preincubated C6 cells, MeHg treatment resulted in a significant (p < 0.001) decrease in GSH levels as compared to coincubation group. Treatment with SeMet did not induce any significant changes in MTT activity in either of the cell lines as compared to the MeHg group. However, the amount of MeHg-induced ROS was significantly reduced (p < 0.001) after SeMet preincubation in both the cell lines. The intracellular Se content was measured with high resolution-inductively coupled plasma mass spectrometry (HR-ICPMS). In both the cell lines the intracellular Se levels increased after pre- and coincubation with 20 and 50 μM SeMet. However, the preincubation group exhibited increased Se content in both the cell lines and varied (p < 0.001) from coincubation group. These differences in the Se content were maintained after 10 μM MeHg treatment for 50 min. In C6-gliomas, the cell associated-MeHg measurements using ¹⁴C-labeled MeHg indicated a significant increase (p < 0.001) in MeHg content in preincubated cells as compared to coincubated cells. These findings provide experimental evidence that preincubation with SeMet increases Se content in cells and prevents against increased MeHg-induced ROS generation.     

Alterations in acetylcholine,PGE2 and IL6 release from urothelial cells following treatment with pyocyanin and lipopolysaccharide

The effects of pseudomonal virulence factor pyocyanin, and LPS from Pseudomonas aeruginosa and Escherichia coli on urothelial mediator release and cytokine production were examined. RT4 urothelial cells were treated with pyocyanin (1–100 μM) or LPS (1–100 ng/mL) for 24-h. Effects were measured in terms of changes in cell viability, basal and stretch-induced acetylcholine (Ach) and PGE₂ release, and inflammatory cytokines (IL-6 and IL-12) production. Twenty-four hour pyocyanin (100 μM) treatment significantly decreased urothelial cell viability, while stretch-induced Ach release response was inhibited. E. coli LPS (100 ng/mL) produced a similar response with an additional significant increase in basal Ach release. All three virulence factors significantly increased urothelial PGE₂ release; under basal release for pyocyanin (100 μM), stretch-induced release for pseudomonal LPS (?10 ng/mL) and both basal and stimulated release for E. coli LPS (?10 ng/mL). IL-6 and IL-12 were not detected in control samples, however 24 h treatment with pyocyanin (100 μM) or LPS (100 ng/mL) resulted in IL-6 release from urothelial cells. The changes in urothelial Ach and PGE₂, and release of inflammatory cytokine IL-6 induced by exposure to the bacterial virulence factors may play a role in the symptoms of pain and urinary urgency experienced with urinary tract infections.     

Sodium selenite and vitamin E in preventing mercuric chloride induced renal toxicity in rats

This study aims to investigate improving effects of sodium selenite and/or vitamin E on mercuric chloride-induced kidney impairments in rats. Wistar male rats were exposed either to sodium selenite (0.25 mg/kg day), vitamin E (100 mg/kg day), sodium selenite + vitamin E, mercuric chloride (1 mg/kg day), sodium selenite + mercuric chloride, vitamin E + mercuric chloride and sodium selenite + vitamin E + mercuric chloride for 4 weeks. Mercuric chloride exposure resulted in an increase in the uric acid, creatinine, blood urea nitrogen and malondialdehyde (MDA) levels and a decrease in the superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities. Histopathological changes were detected in kidney tissues in mercuric chloride-treated groups. A significant decrease in the uric acid, creatinine, blood urea nitrogen and MDA levels and a significant increase in the SOD, CAT and GPx activities were observed in the supplementation of sodium selenite and/or vitamin E to mercuric chloride-treated groups.Conclusively, sodium selenite, vitamin E and vitamin E + sodium selenite significantly reduce mercuric chloride induced nephrotoxicity in rats, but not protect completely.     

Increased aquaglyceroporin 9 expression disrupts arsenic resistance in human lung cancer cells

Resistance to chemotherapy is one of the major problems in treatment responses of lung cancer. This study explored the mechanism underlying the arsenic resistance of lung cancer. Four lung cancer cells with different proliferation activity were characterized for cytotoxicity, arsenic influx/efflux, and arsenic effects on intracellular glutathione and 8-hydroxy-2′-deoxyguanosine (8-OHdG) production. Our data revealed that relative proliferation potency of these cells was H1299 > A549 > CL3 > H1355. Moreover, A549, H1299, and H1355 were markedly resistant to As₂O₃ with IC50 ≈ 100 μM, whereas CL3 was sensitive to As₂O₃ with IC50 ≈ 11.8 μM. After treatment with the respective As₂O₃ at IC50, arsenic influx/efflux activity in CL3 was comparable to those in the other three arsenic-resistant cells. However, differences in glutathione levels and 8-OHdG production were also detected either before or after arsenic treatment, indicating that a certain degree of variation in anti-oxidative systems and/or 8-OHdG repair activity existed in these cell lines. By transfection of an aquaglyceroporin 9 (AQP9) gene, we showed that increased AQP9 expression significantly enhanced arsenic uptake and disrupted arsenic resistance of A549. The present study strongly suggests that membrane transporters responsible for arsenic uptake, such as AQP9, may play a critical role in development of arsenic resistance in human lung cancer cells.     

Evaluation of in vitro cytotoxicity of one-dimensional chain [Fe(salen)(L)]n complexes against human cancer cell lines

The 1d-polymeric iron(III) complexes [Fe(salen)(μ-L)]_n (1–6), involving a deprotonated form of the N-donor heterocyclic compounds (L) imidazole (complex 1), 1,2,4-triazole (2), benztriazole (3), 5-methyltetrazole (4), 5-aminotetrazole (5) and 5-phenyltetrazole (6), were studied for their in vitro cytotoxic activity against human cancer cell lines including lung carcinoma (A549), cervix epithelial carcinoma (HeLa), osteosarcoma (HOS), malignant melanoma (G361), breast adenocarcinoma (MCF7), ovarian carcinoma (A2780) and cisplatin-resistant ovarian carcinoma (A2780cis). Cytotoxicity in vitro (IC₅₀ = 0.39–0.48 μM) was achieved for 2–6 against A2780 (IC₅₀ of cisplatin equals 11.5 μM) as well as for 5 and 6 against all the tested cells, with IC₅₀ = 2.5–37.7 μM. The Uv–Vis spectroscopic study showed that the complexes are unstable in organic solvents (e.g. dimethyl sulfoxide, dimethylformamide) containing even trace amounts of water (and thus also in the medium, i.e. 0.1% DMF, v/v, used in the MTT assay), where they partially or completely decompose to the mixtures involving, besides [Fe(salen)(μ-L)]_n itself, also the starting compounds [{Fe(salen)}₂(μ-O)] and appropriate organic compound (HL). In efforts to find how the resulting cytotoxicity of the most active compounds 5 and 6 is influenced by this fact, the in vitro cytotoxicity testing of mixtures of reactants [{Fe(salen)}₂(μ-O)] and HL, as well as the respective reactants, was also performed. It has been found that the cytotoxicity of 5 and 6 against all the tested cell lines is probably caused by a combined effect of the individual components presented within the corresponding mixture in the medium used.