Oxidative stress contributes to gold nanoparticle-induced cytotoxicity in human tumor cells

Due to their exceptional properties, gold nanoparticles (AuNPs) have shown promising medical and technological applications in the treatment of cancer and the development of antimicrobial packaging and time–temperature indicators in the food sector. However, little is known about their cytotoxicity when they come into contact with biological systems. The aim of this work was to compare the effects of three commercially available AuNPs of different sizes (30, 50 and 90?nm) on human leukemia (HL-60) and hepatoma (HepG2) cell lines. AuNP-induced cytotoxicity was dose and time-dependent, with IC₅₀ values higher than 15?μg/mL. Nanoparticle (NP) size and cell line slightly influenced on the cytotoxicity of AuNPs, although HL-60 cells proved to be more sensitive to the cytotoxic response than HepG2. N-Acetyl-l-cysteine (NAC) protected HL-60 and HepG2 cells only against treatment with 30?nm AuNPs. In both cell types, glutathione (GSH) content was drastically depleted after 72?h of incubation with the three AuNPs (less than 30% in all cases), while the reduction of superoxide dismutase activity (SOD) activity depended on cell line. HepG2, but not HL-60 cells, exhibited a decrease of SOD activity (～45% of activity). The three AuNPs also caused a two-fold elevation of reactive oxygen species (ROS) production in both cell lines. Thus, protective effect of NAC, depletion of GSH and increase of ROS appear to be determined by NP size and indicate that oxidative stress contributes to cytotoxicity of AuNPs.     

Formation of a 3,4-diol-1,2-epoxide metabolite of benz[a]anthracene with cytotoxicity and genotoxicity in a human in vitro hepatocyte culture system

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants that require metabolic activation to exert their carcinogenic effects. This study investigated the 3,4-diol-1,2-epoxide formation of benz[a]anthracene (BA) and its toxic effects in a human in vitro hepatocyte culture system. Both mRNA and protein expression of metabolic enzymes which can activate PAHs to carcinogenic forms increased after BA exposure in HepG2 cells and our quantitative analysis showed that the formation of BA-3,4-diol-1,2-epoxide in medium extracts increased in a time-dependent manner. We also performed several comparative studies which show that much lower concentrations of BA-3,4-diol-1,2-epoxide had stronger cytotoxicity and genotoxicity than higher doses of BA. These results suggest that BA is activated as the major carcinogenic metabolite 3,4-diol-1,2-epoxide, in human in vitro culture systems by metabolic enzymes and that this metabolite has stronger cytotoxic and genotoxic effects than its parent compound.     

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.     

Oligomeric proanthocyanidins alleviate hexabromocyclododecane-induced cytotoxicity in HepG2 cells through regulation on ROS formation and mitochondrial pathway

Hexabromocyclododecane (HBCD), a type of brominated flame retardants (BFR), has become ubiquitous organic contaminants in recent years. However, studies on HBCD toxicity and the related molecular mechanisms are so far limited. The objective of the present study was to investigate the effects of oligomeric proanthocyanidins (OPCs) on cytotoxicity induced by HBCD and the underlying molecular mechanisms. HepG2 cells were treated with HBCD and/or OPCs at different concentrations, and cell viability, cell apoptosis, reactive oxygen species (ROS) production, cellular Ca²⁺ level, mitochondrial membrane potential (ΔΨ), cytochrome C (Cyt-c) release, and nuclear factor-erythroid 2-related factor 2 (Nrf2) proteins expression were evaluated. Results showed that HBCD induced toxic effects in HepG2 cells in a concentration-dependent manner. HBCD at high concentrations (40 and 60 μM) caused a significant decrease of cell viability and led to elevated cell apoptosis ratio, intracellular Ca²⁺ level, cytoplasmic Cyt-c level, and ROS production, together with a loss of ΔΨ and mobilization of Nrf2. Pretreatment with OPCs effectively attenuated the cytotoxic effects and ROS production, as well as mitochondrial responses induced by HBCD. Thus, OPCs could alleviate cytotoxicity in HepG2 cells induced by HBCD through regulation on intracellular Ca²⁺ level and ROS formation in a mitochondrial pathway.     

Cytotoxicity of 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione (DCPT) and analogues in wild type and CYP3A4 stably transfected HepG2 cells

The thiazolidinedione (TZD) ring is a constituent of the glitazones that are used to treat type II diabetes. Liver injury has been reported following chronic glitazone use; however, they do not produce hepatic damage in common laboratory animal species. In contrast, 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione (DCPT) causes hepatotoxicity in rats. DCPT toxicity is dependent upon the presence of an intact TZD ring and cytochrome P450 (CYP)-mediated biotransformation. To further investigate TZD ring-induced toxicity, DCPT and several structural analogues or potential metabolites were tested in vitro using wild type human hepatoma HepG2 and HepG2 cells stably transfected with the CYP3A4 isozyme. CYP3A4 activity was confirmed by measuring testosterone 6β-hydroxylation. Both cell lines were treated with 0-250 μM of the compounds in Hanks' balanced salt solution. Cell viability was measured after 24 h. DCPT and S-(3,5-dichlorophenyl)aminocarbonyl thioglycolic acid (DCTA) were the most toxic compounds of the series. Furthermore, DCPT was significantly more toxic in transfected cells (LC50=160.2±5.9 μM) than in wild type cells (LC50=233.0±19.7 μM). Treatment with a CYP3A4 inhibitor or inducer attenuated or potentiated DCPT cytotoxicity, respectively. These results suggest that DCPT-induced cytotoxicity in the transfected HepG2 cells is partially dependent on CYP3A4.     

Toxicity of cholesterol oxidation products to Caco-2 and HepG2 cells: modulatory effects of alpha- and gamma-tocopherol

Cholesterol can be oxidized to form a variety of cholesterol oxidation products also known as oxysterols. The aims of the present study were to compare the cytotoxic effects of four oxysterols, namely 25-hydroxycholesterol (25-OHC), 7beta-hydroxycholesterol (7beta-OHC), cholesterol-5beta,6beta-epoxide (beta-epox) and cholesterol-5alpha,6alpha-epoxide (alpha-epox), in two human cell culture models. Further, the ability of 10 and 100 micro m alpha- and gamma-tocopherol (alpha-TOC and gamma-TOC, respectively) to protect against oxysterol-induced cytotoxicity was also assessed. Human colonic adenocarcinoma Caco-2 and human hepatoma HepG2 cells were supplemented with increasing concentrations of 25-OHC, 7beta-OHC, beta-epox and alpha-epox (0-25 micro g ml(-1)) for 24, 48 or 96 h. Following 24-h and 48-h exposure, test media were replaced with normal growth media and the cells were maintained for 72 and 48 h, respectively. The 96-h exposure represented a constant challenge to the cells. Cytotoxicity was assessed using the neutral red uptake assay. The concentration of compound that inhibited cell viability by 50% (ic(50) value) was calculated. All four oxysterols investigated induced the greatest cytotoxic effects following 96 h of exposure. 25-Hydroxycholesterol exhibited the greatest cytotoxicity in both cell lines. Both beta-epox and alpha-epox were more toxic to HepG2 cells than to Caco-2 cells after the 48-h exposure. Pretreatment of cells with either alpha- or gamma-TOC did not protect against oxysterol-induced cytotoxicity. The caco-2 cells treated with the high concentration (100 micro m) of gamma-TOC were found to be more susceptible to oxysterol-induced toxicity under the conditions employed in this study.     

Involvement of enniatins-induced cytotoxicity in human HepG2 cells

Enniatins (ENNs) are mycotoxins found in Fusarium fungi and they appear in nature as mixtures of cyclic depsipeptides. The ability to form ionophores in the cell membrane is related to their cytotoxicity. Changes in ion distribution between inner and outer phases of the mitochondria affect to their metabolism, proton gradient, and chemiosmotic coupling, so a mitochondrial toxicity analysis of enniatins is highly recommended because they host the homeostasis required for cellular survival. Two ENNs, ENN A and ENN B on hepatocarcinoma cells (HepG2) at 1.5 and 3 μM and three exposure times (24, 48 and 72 h) were studied. Flow cytometry was used to examine their effects on cell proliferation, to characterize at which phase of the cell cycle progression the cells were blocked and to study the role of the mitochondrial in ENNs-induced apoptosis. In conclusion, apoptosis induction on HepG2 cells allowed to compare cytotoxic effects caused by both ENNs, A and B. It is reported the possible mechanism observed in MMP changes, cell cycle analysis and apoptosis/necrosis, identifying ENN B more toxic than ENN A.     

Cytotoxicity and QSAR study of (thio)ureas derived from phenylalkylamines and pyridylalkylamines

Simplified 1,3-disubstituted urea derivatives (11–24) of phenylethylamines, homoveratylamines, 2-pyridylethylamines, 2-picolylamines as well as xylylenediamines were synthesized and investigated for their cytotoxic activities. The results revealed that most analogs displayed cytotoxicity against HepG2 and MOLT-3 cell lines. The bis-thiourea derivatives 23 and 24 exhibited higher inhibitory potency against HepG2 cell than the reference drug, etoposide. 1,1′-(1,3-phenylenebis(methylene))bis(3-(4-chlorophenyl)thiourea) 24 was shown to be the most potent cytotoxic compound against MOLT-3 cell line with an IC₅₀ value of 1.62 μM. QSAR studies suggested that compounds with high ionization potential displayed high cytotoxicity against HuCCA-1 cell line. Furthermore, derivatives with dimethoxyphenyl group had high radial distribution function with a correspondingly high cytotoxicity against A549 cell line. Moreover, analogs 23 and 24 had low values of E _HOMO (energy of the highest occupied molecular orbital energy) as well as high cytotoxicity against HepG2 cell line. This study affords an easily accessible approach for the synthesis of promising anticancer agents. The developed QSAR models provided pertinent information into the physicochemical properties governing the investigated biologic properties.     

Role of metabolism by human intestinal microflora in geniposide-induced toxicity in HepG2 cells

Possible role of metabolism by the intestinal bacteria in geniposide-induced cytotoxicity was investigated in human hepatoma HepG2 cells. Initially, toxic effects of geniposide and its metabolite genipin were compared. Genipin, a deglycosylated form of geniposide, was cytotoxic at the concentrations that geniposide was not. As metabolic activation systems for geniposide, human intestinal bacterial cultures, fecal preparation (fecalase) and intestinal microbial enzyme mix were employed in the present study. When geniposide was incubated with human intestinal bacteria, either Bifidobacterium longum HY8001 or Bacteroides fragilis, for 24 h, the cultured media caused cytotoxicity in HepG2 cells. Fecalase and intestinal enzyme mix were also effective to metabolically activate geniposide to its cytotoxic metabolite. The present results indicated that genipin, a metabolite of geniposide, might be more toxic than geniposide, and that intestinal bacteria might have a role in biotransformation of geniposide to its toxic metabolite. In addition, among three activation systems tested, intestinal microbial enzyme mix would be convenient to use in detecting toxicants requiring metabolic activation by intestinal bacteria.     

The accumulation and toxicity of methylated arsenicals in endothelial cells: important roles of thiol compounds

Excess intake of arsenic is known to cause vascular diseases as well as skin lesions and cancer in humans. Recent reports suggest that trivalent methylated arsenicals, which are intermediate metabolites in the methylation process of inorganic arsenic, are responsible for the toxicity and carcinogenicity of environmental arsenic. We investigated acute toxicity and accumulation of monomethylarsonic acid (MMA(V)), dimethylarsinic acid (DMA(V)), trimethylarsine oxide (TMAO), and monomethylarsonous acid diglutathione (MMA(III) (GS)(2)) in rat heart microvessel endothelial (RHMVE) cells. MMA(V) (LC(50) = 36.6 mM) and DMA(V) (LC(50) = 2.54 mM) were less toxic than inorganic arsenicals (cf. LC(50) values for inorganic arsenite (iAs(III)), and inorganic arsenate (iAs(V)) was reported to be 36 and 220 microM, respectively, in RHMVE cells. TMAO was essentially not toxic. However, MMA(III) (GS)(2) was highly toxic (LC(50) = 4.1 microM). The order of cellular arsenic accumulation of those four organic arsenic compounds was MMA(III) (GS)(2) > MMA(V) > DMA(V) > TMAO. MMA(III) (GS)(2) was efficiently taken up by the cells and cellular arsenic content increased with the concentration of MMA(III) (GS)(2) in culture medium. N-acetyl-l-cysteine (NAC) reduced cellular arsenic content in DMA(V)-exposed cells and also decreased the cytotoxicity of DMA(V), whereas it changed neither cellular arsenic content nor the viability in MMA(V)-exposed cells. mRNA levels of heme oxygenase-1 (HO-1) were decreased by NAC in DMA(V)-exposed, but MMA(V)-exposed cells. Buthionine sulfoximine (BSO), a cellular glutathione (GSH) depleting agent, enhanced the cytotoxicity of MMA(V). However, BSO reduced, rather than enhanced, the cytotoxicity of DMA(V). These results suggest that intracellular GSH modulated the toxic effects of arsenic in opposite ways for MMA(V) and DMA(V). Even though intracellular GSH decreased the cytotoxicity of MMA(V), extracellularly added GSH enhanced the cytotoxicity of MMA(V). The use of high-performance liquid chromatography (HPLC)-inductively coupled plasma mass spectrometric analyses suggested that a small amount of MMA(V) was converted to MMA(III) (GS)(2) in the presence of GSH. These results suggest that MMA(III) (GS)(2) is highly toxic compared to other arsenic compounds because of faster accumulation of this species by cells, in addition to having the toxic nature of methylated trivalent organic arsenics.     

Effects of in vitro exposures to cadmium, mercury, zinc, and 1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2-dichloroethane on steroidogenesis by dispersed interrenal cells of rainbow trout (Oncorhynchus mykiss).

Numerous environmental xenobiotics can act as endocrine disrupters in wildlife species. Fish chronically exposed to pollutants exhibit a deficiency in the synthesis of cortisol, a glucocorticosteroid hormone secreted by interrenal steroidogenic cells in response to ACTH by a mechanism mediated by cAMP. The capacity of a series of heavy metals (CdCl2, ZnCl2, HgCl2, and CH3HgCl) and 1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2-dichloroethane (o,p'-DDD) to disrupt cortisol secretion was determined in dispersed interrenal cells of rainbow trout (Oncorhynchus mykiss) exposed in vitro to the toxicant, by measuring cortisol secretion stimulated with ACTH or dibutiryl-cAMP (dbcAMP) and by assessing cell viability. The effect of cadmium in presence of zinc on the interrenal cells was also determined. The median lethal concentration (LC50, dose that kills 50% of the cells), median effective concentration (EC50, dose that inhibits cortisol secretion by 50%), and LC50/EC50 ratio were determined for each chemical to compare its endocrine toxicity and to test the specificity of the toxicants to act as endocrine disrupters. HgCl2 had the lowest EC50 and LC50; it was the most toxic of the chemicals tested. The LC50/EC50 ratio was the highest for ZnCl2 and CdCl2, indicating that these toxicants had the most specific endocrine toxicity. The mechanism of toxicity of heavy metals on cortisol-secreting cells involves a site situated downstream from the step generating cAMP, while o,p'-DDD seemed to impair a step located between adenyl cyclase activation and the ACTH binding. No evidence for a protector effect of zinc against cadmium toxicity was found.     

Primary cultures of rabbit renal proximal tubule cells. III. Comparative cytotoxicity of inorganic and organic mercury.

The present study further developed primary cultures of rabbit renal proximal tubule cells (RPTC) as an in vitro model to study chemical-induced toxicity by investigating the comparative cytotoxicity of mercuric chloride (HgCl2) and methyl mercury chloride (CH3HgCl) to RPTC. Confluent monolayer cultures of RPTC exposed to HgCl2 and CH3HgCl for 24 hr exhibited a concentration-dependent loss in cell viability at culture medium concentrations greater than 25 and 2.5 microM, respectively. Vital dye exclusion was a more sensitive indicator of cytotoxicity than the amount of lactate dehydrogenase activity, alkaline phosphatase activity, N-acetylglucosaminidase activity, and protein content remaining on the culture dish. On the basis of vital dye exclusion, HgCl2 was less toxic to proximal tubule cells in culture than CH3HgCl after 24 hr of exposure, whether cytotoxicity was based on LC50 values (34.2 microM HgCl2 vs 6.1 microM CH3HgCl) or total cellular mercury uptake (4.6 nmol Hg2+/10(5) cells vs 1.25 nmol CH3Hg+/10(5) cells). Differences in the extent and rate of metal uptake were also evident. Maximum cellular uptake of Hg2+ occurred within 6-24 hr after exposure and was not concentration-dependent, whereas maximum uptake of CH3Hg+ occurred within 3 hr of exposure and was concentration-dependent. The intracellular distribution of both mercurials between acid-soluble and acid-insoluble binding sites also differed. At noncytotoxic concentrations of HgCl2 (0.04-5 microM), intracellular Hg2+ bound increasingly to acid-soluble binding sites as a function of time, from 15-30% after 6 hr of exposure to 40-60% after 72 hr of exposure. However, at subcytotoxic (25 microM) and cytotoxic (34.2 microM) concentrations, Hg2+ binding to acid-soluble binding sites remained constant at approximately 30-40% for 6, 12, 24, and 72 hr after exposure. In contrast, only 20% of total cellular CH3Hg+ was bound to acid-soluble binding sites after exposure to 0.039 to 6.1 microM CH3HgCl for 6, 12, and 24 hr. Total cellular glutathione content was unaffected after exposure to 0.04-5 microM HgCl2 and 0.039-6.1 microM CH3HgCl, but was depleted 6 hr after exposure to 25 and 34.2 microM HgCl2. These results indicate that CH3HgCl was a more potent cytotoxicant to RPTC in primary culture than HgCl2. Furthermore, compared to Hg2+, the low binding of CH3Hg+ to acid-soluble binding sites and the absence of a redistribution of CH3Hg+ from acid-insoluble to acid-soluble binding sites appeared to contribute to its more potent toxicity to cultured cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chromone linked nitrone derivative induces the expression of iNOS2 and Th1 cytokines but reduces the Th2 response in experimental visceral leishmaniasis

In our previous work we have shown that the novel synthetic chromone derivative could effectively inhibit the Leishmania donovani replication in vitro and in vivo with less cytotoxicity on murine splenocytes. The aim of the present study is to explore the possible mechanism of anti-leishmanial effect of C-(6-methyl-4-oxo-4H-1-benzopyran-3-yl)-N-(p-tolyl) nitrone (designated as NP1) in vitro and in vivo in experimental visceral leishmaniasis caused by L. donovani. The cytotoxic effect of this derivative was studied in murine peritoneal macrophages by MTT method. NP1 at a dose of 17.06 μM showed 50% inhibition on L. donovani promastigotes but found less cytotoxic to the RAW 264.7 cells. Even the higher concentration of IC50 (up to four fold) did not exert much cytotoxic effect on RAW 264.7. Interestingly, NP1 at lower concentration (8.53 μM) could inhibit 50% of intracellular amastigotes in murine peritoneal macrophages. L. donovani is known to exert its pathogenic effects mainly by the suppression of NO generation and subversion of the cellular inflammatory responses in the macrophages. NP1 was found to induce a potent host-protective immune response by enhancing NO generation and iNOS2 expression at mRNA level and by up-regulating proinflammatory cytokines such as IL-12 and IFN-γ and limiting the expression of IL-10 in vivo. The NO dependent killing was further confirmed in iNOS^?/? mice compared to wild type. In agreement with the fact, induced synthesis of IL-12 and IFN-γ and associated down-regulation of IL-10 by the treatment of NP1 clearly indicated the possibility of novel strategy of drug development against Leishmania infection.     

Cytotoxicity of chloroacetanilide herbicide alachlor in HepG2 cells independent of CYP3A4 and CYP3A7.

Alachlor is cytotoxic to human hepatoblastoma HepG2s, a cell line that expresses constitutive CYP3A7 and dexamethasone (DEX)-inducible CYP3A4 and CYP3A7. CYP3A4 catalyzes alachlor N-dealkylation to 2-chloro-N-(2,6-diethylphenyl)acetamide (CDEPA), precursor of 2,6-diethylbenzoquinoneimine, putative reactive metabolite for rat nasal carcinogenicity. We hypothesized that HepG2 alachlor cytotoxicity would be mediated by CYP3A4/7 and increased with DEX. Here, we report time-dependent alachlor cytotoxicity (EC(50) approximately 500 microM and 264+/-17 microM at 6 and 24h, respectively) as assessed by lactate dehydrogenase leakage. DEX pretreatment (25 microM, 48 h) significantly increased CYP3A7-catalyzed luciferin 6' benzylether O-debenzylation, but had no effect on alachlor toxicity. Further, CYP3A4/7 inhibitor triacetyloleandomycin did not prevent, but rather potentiated, alachlor cytotoxicity. In agreement, CDEPA was less toxic than parent alachlor. HepG2 CYP3A4 activity was unaffected by 48 h DEX pretreatment; therefore, studies were done in DPX-2 cells, a HepG2 derivative engineered to overexpress pregnane-X receptor (PXR) that exhibits rifampicin (RIF)-inducible endogenous CYP3A4. Alachlor cytotoxicity in DPX-2 cells occurred over a concentration range equivalent to that in HepG2. CYP3A4 activity of DPX-2 cells treated with RIF (10 microM, 48 h) was twice that of untreated cells, but RIF did not increase alachlor toxicity. These results demonstrate that neither CYP3A4 nor CYP3A7 initiate a pathway leading to a toxic alachlor metabolite.     

Dibenzofuran induces oxidative stress,disruption of trans-mitochondrial membrane potential (ΔΨm) and G1 arrest in human hepatoma cell line

Dioxins are a class of extremely toxic environmentally persistent pollutant, comprised of halogenated dibenzo-p-dioxins, dibenzofurans and biphenyls. Despite significant human exposure via multiple routes, very little is known about toxicity induced by dibenzofuran (DF). Current study shed lights on the potential toxicity mechanism of DF using human hepatoma cell line (HepG2). It was observed that the exposure to DF potentiate oxidative stress, apoptosis and necrosis at 10 μM within 8 h in HepG2 cells. Interestingly, when we pre-incubated the cells with α-NF (1 nM) for 12 h, an aromatic hydrocarbon receptor antagonist, the IC₅₀ of DF increased by 14 folds indicating the cytoprotective ability of α-NF from DF induced toxicity. Furthermore, three additional metabolites were observed while studying the metabolic profile of DF in HepG2 cells with and without pre-incubation with α-NF using chromatography–mass spectroscopy (GC–MS). Of these, two metabolites were characterized as dihydroxylated derivative of DF and third metabolite was characterized as quinone derivative of DF. By flow cytometry and confocal laser microscopy analysis we followed the ROS formation after DF (10 μM) exposure for 3 h. Significantly low ROS was generated in cells which were pre-incubated with α-NF than cells which were not pre-incubated with α-NF underlining the importance of metabolism in DF toxicity. The same pattern of protection was consistent while measuring mitochondrial membrane potential (MMP), i.e., less MMP dip was observed in ‘with α-NF pre-incubated and DF (10 μM) exposed cells’ than ‘without α-NF pre-incubated but DF exposed cells’. In cell cycle studies, it was confirmed that cell population of HepG2 at G1 stage progressively increased in number (∼74%) within 24 h. Thus, DF and its metabolites induce significantly higher cytotoxicity after metabolism in HepG2 cells than its parent compound (DF) by ROS formation, MMP dip and impaired cell cycle.     

Synthesis of flurbiprofen thiadiazole urea derivatives and assessment of biological activities and molecular docking studies

Totally 15 novel flurbiprofen urea derivatives were synthesized bearing the thiadiazole ring. Their inhibition effects on tyrosinase were determined. 3c was found to be the strongest inhibitor with the IC₅₀ value of 68.0 μM against tyrosinase. The enzyme inhibition types of the synthesized compounds were determined by examining the kinetic parameters. The inhibition type of 3c was determined as uncompetitive and the K_i value was calculated as 36.3 μM. Moreover, their cytotoxic effects on hepatocellular carcinoma (HepG2), colorectal carcinoma (HT-29), and melanoma (B16F10) cell lines were evaluated. According to the cytotoxicity results, 3l (IC₅₀ = 14.11 μM) showed the highest cytotoxicity on the HT-29 cells, while 3o (IC₅₀ = 4.22 μM) exhibited the strongest cytotoxic effect on HepG2 cell lines. Also, 3j (IC₅₀ = 7.55 μM strongly affected B16F10. The effects of synthesized compounds on the healthy cell line were evaluated on the CCD-986Sk cell line. Molecular modelling studies have indicated the potential binding interactions of the uncompetitive inhibitor 3c with the enzyme-substrate complex.