V79 Chinese Hamster Lung Fibroblasts Research Articles

Mechanism of action studies of lomaiviticin A and the monomeric lomaiviticin aglycon. Selective and potent activity toward DNA double-strand break repair-deficient cell lines.

(-)-Lomaiviticin A (1) and the monomeric lomaiviticin aglycon [aka: (-)-MK7-206, (3)] are cytotoxic agents that induce double-strand breaks (DSBs) in DNA. Here we elucidate the cellular responses to these agents and identify synthetic lethal interactions with specific DNA repair factors. Toward this end, we first characterized the kinetics of DNA damage by 1 and 3 in human chronic myelogenous leukemia (K562) cells. DSBs are rapidly induced by 3, reaching a maximum at 15 min post addition and are resolved within 4 h. By comparison, DSB production by 1 requires 2-4 h to achieve maximal values and >8 h to achieve resolution. As evidenced by an alkaline comet unwinding assay, 3 induces extensive DNA damage, suggesting that the observed DSBs arise from closely spaced single-strand breaks (SSBs). Both 1 and 3 induce ataxia telangiectasia mutated- (ATM-) and DNA-dependent protein kinase- (DNA-PK-) dependent production of phospho-SER139-histone H2AX (γH2AX) and generation of p53 binding protein 1 (53BP1) foci in K562 cells within 1 h of exposure, which is indicative of activation of nonhomologous end joining (NHEJ) and homologous recombination (HR) repair. Both compounds also lead to ataxia telangiectasia and Rad3-related- (ATR-) dependent production of γH2AX at later time points (6 h post addition), which is indicative of replication stress. 3 is also shown to induce apoptosis. In accord with these data, 1 and 3 were found to be synthetic lethal with certain mutations in DNA DSB repair. 1 potently inhibits the growth of breast cancer type 2, early onset- (BRCA2-) deficient V79 Chinese hamster lung fibroblast cell line derivative (VC8), and phosphatase and tensin homologue deleted on chromosome ten- (PTEN-) deficient human glioblastoma (U251) cell lines, with LC50 values of 1.5 ± 0.5 and 2.0 ± 0.6 pM, respectively, and selectivities of >11.6 versus the isogenic cell lines transfected with and expressing functional BRCA2 and PTEN genes. 3 inhibits the growth of the same cell lines with LC50 values of 6.0 ± 0.5 and 11 ± 4 nM and selectivities of 84 and 5.1, for the BRCA2 and PTEN mutants, respectively. These data argue for the evaluation of these agents as treatments for tumors that are deficient in BRCA2 and PTEN, among other DSB repair factors.

The major DNA repair pathway after both proton and carbon-ion radiation is NHEJ, but the HR pathway is more relevant in carbon ions.

The purpose of this study was to identify the roles of non-homologous end-joining (NHEJ) or homologous recombination (HR) pathways in repairing DNA double-strand breaks (DSBs) induced by exposure to high-energy protons and carbon ions (C ions) versus gamma rays in Chinese hamster cells. Two Chinese hamster cell lines, ovary AA8 and lung fibroblast V79, as well as various mutant sublines lacking DNA-PKcs (V3), X-ray repair cross-complementing protein-4 [XRCC4 (XR1), XRCC3 (irs1SF) and XRCC2 (irs1)] were exposed to gamma rays ((137)Cs), protons (200 MeV; 2.2 keV/μm) and C ions (290 MeV; 50 keV/μm). V3 and XR1 cells lack the NHEJ pathway, whereas irs1 and irs1SF cells lack the HR pathway. After each exposure, survival was measured using a clonogenic survival assay, in situ DSB induction was evaluated by immunocytochemical analysis of histone H2AX phosphorylation at serine 139 (γ-H2AX foci) and chromosome aberrations were examined using solid staining. The findings from this study showed that clonogenic survival clearly depended on the NHEJ and HR pathway statuses, and that the DNA-PKcs(-/-) cells (V3) were the most sensitive to all radiation types. While protons and γ rays yielded almost the same biological effects, C-ion exposure greatly enhanced the sensitivity of wild-type and HR-deficient cells. However, no significant enhancement of sensitivity in cell killing was seen after C-ion irradiation of NHEJ deficient cells. Decreases in the number of γ-H2AX foci after irradiation occurred more slowly in the NHEJ deficient cells. In particular, V3 cells had the highest number of residual γ-H2AX foci at 24 h after C-ion irradiation. Chromosomal aberrations were significantly higher in both the NHEJ- and HR-deficient cell lines than in wild-type cell lines in response to all radiation types. Protons and gamma rays induced the same aberration levels in each cell line, whereas C ions introduced higher but not significantly different aberration levels. Our results suggest that the NHEJ pathway plays an important role in repairing DSBs induced by both clinical proton and C-ion beams. Furthermore, in C ions the HR pathway appears to be involved in the repair of DSBs to a greater extent compared to gamma rays and protons.

Alteration in the Doxorubicin-Induced DNA Damage in Cultured V79 Cells by Aegle marmelos (L.) Correa (Bael): A Comet Assay Study

The clinical applicability of wide spectrum chemotherapeutic drug, doxorubicin is limited due to induction of severe cardiomyopathy resulting from DNA damage. The present study was aimed to evaluate the protective effect of bael (Aegle marmelos) extract (AME) on the doxorubicin-induced molecular DNA damage in V79 cells. V79 (Chinese hamster lung fibroblasts) cells were treated with 0 or 25 μg/ml AME before exposure to 0, 1, 2.5, 5, 10, 25 or 50 μg/ml doxorubicin DOX. The DNA damage was studied at different post-doxorubicin treatment times using single cell gel electrophoresis. Doxorubicin caused a maximum DNA damage at 1 h post-DOX treatment indicated by a highest Olive Tail Movement (OTM) and tail DNA, whereas treatment of V79 cells with 25 μg/ml AME enhanced DNA repair at all assessment times with a maximum repair up to 6 h which did not alter thereafter. In another experiment DOX caused a concentration dependent increase in the DNA damage and treatment of V79 cells with 25 μg/ml AME significantly inhibited DOX-induced DNA damage at all post-DOX treatment times. The rate of DNA repair was higher in AME pre-treated cells than DOX-treatment alone. Assessment of cell survival showed a concentration dependent decline in the clonogenicity after DOX-treatment, whereas AME pre-treatment arrested the DOX-induced reduction in the cell survival. The DNA damage and clonogenicity of cells showed a close but inverse relationship, i.e., with increasing DNA damage there was a corresponding reduction in the cell survival. This relationship between cell survival and DNA damage was linear quadratic. Our study demonstrates that AME pretreatment reduced the DOX-induced DNA damage and hastened the DNA repair in V79 cells, thus demonstrating the chemoprotective potential of AME.

<i>In Vitro</i> Safety Evaluation of Nanomaterials<br/>—Cellular Response to Metal Oxide Nanoparticles—

Although nanomaterials are already being used for various applications in the industry, the safety of nanomaterials has not yet been sufficiently elucidated. An in vitro cellular toxicological study using well-characterized nanomaterials is conducted for the evaluation of the biological effects of nanomaterials. In this study, the effects of copper oxide nanoparticles on the global gene expression of human lung epithelial A549 cells were analyzed, and the molecular responses of A549 cells to the toxicity of the copper oxide (CuO) nanoparticles were inferred. Furthermore, the cytotoxic effects of silicon dioxide (SiO2) nanoparticles and titanium dioxide (TiO2) nanoparticles coexisting with some metal salts in Chinese hamster lung fibroblast V79 cells were also examined, and SiO2 nanoparticles increased the cytotoxicity of some of the coexisting metal salts as a result. Finally, the importance of in vitro studies in the safety evaluation of nanomaterials was discussed.

Monitoring of potential cytotoxic and inhibitory effects of titanium dioxide using on-line and non-invasive cell-based impedance spectroscopy

Titanium dioxide (TiO2) nanoparticles (NPs) with different sizes and structures were probed for plausible cytotoxicity using electric cell-substrate impedance sensing (ECIS), a non-invasive and on-line procedure for continuous monitoring of cytotoxicity. For insect cells (Spodoptera frugiperda Sf9), the ECIS50 values, i.e., the concentration required to achieve 50% inhibition of the response, differed depending on the size and shape of the TiO2 nanostructure. The lowest ECIS50 value (158ppm) was observed for the needle shaped rutile TiO2 (10nm×40nm, 15.5nm nominal particle size), followed by 211ppm for P-25 (34.1nm, 80% anatase and 20% rutile), 302ppm for MTI5 (5.9nm, 99% anatase) and 417ppm for Hombitan LW-S bulk TiO2 (169.5nm, 99% anatase). Exposure of TiO2 NPs to UV light at 254nm or 365nm exhibited no significant effect on the ECIS50 value due to the aggregation of TiO2 NPs with diminishing photocatalytic activities. Chinese hamster lung fibroblast V79 cells, exhibited no significant cytotoxicity/inhibition up to 400ppm with P25, MTI5 and bulk TiO2. However, a noticeable inhibitory effect was observed (ECIS50 value of 251ppm) with rutile TiO2 as cell spreading on the electrode surface was prevented

Probing inhibitory effects of nanocrystalline cellulose: inhibition versus surface charge

NCC derived from different biomass sources was probed for its plausible cytotoxicity by electric cell-substrate impedance sensing (ECIS). Two different cell lines, Spodoptera frugiperda Sf9 insect cells and Chinese hamster lung fibroblast V79, were exposed to NCC and their spreading and viability were monitored and quantified by ECIS. Based on the 50%-inhibition concentration (ECIS(50)), none of the NCC produced was judged to have any significant cytotoxicity on these two cell lines. However, NCC derived from flax exhibited the most pronounced inhibition on Sf9 compared to hemp and cellulose powder. NCCs from flax and hemp pre-treated with pectate lyase were also less inhibitory than NCCs prepared from untreated flax and hemp. Results also suggested a correlation between the inhibitory effect and the carboxylic acid contents on the NCC.

Indium chloride‐induced micronuclei via reactive oxygen species in Chinese hamster lung fibroblast V79 cells

We study the cytotoxicity of indium chloride (InCl₃) in Chinese hamster lung fibroblasts, the V79 cells, using MTT assay. The results showed that InCl₃ did not induce significant cytotoxicity at various concentrations tested. In addition, the frequency of micronuclei (MN) was assayed to evaluate the genotoxic effects of InCl₃ in V79 cells. InCl₃ at concentrations ranged 0.1-1 μM significantly increased MN frequency in a concentration-dependent manner. Both catalase and superoxide dismutase at concentrations of 75 and 150 μg/mL significantly inhibited InCl₃-induced MN. Similarly, Germanium oxide (GeO₂) and dimercaprol expressed antigenotoxic effects. From these findings, it is concluded that InCl₃ is a potent genotoxic chemical, which may be mediated partly by inducing oxidative stress. The significance of this study shows that the workers in the semiconductor factories should be cautious in exposing to the hazardous genotoxic InCl₃.

Eukaryotic cell lines as a sensitive layer for direct monitoring of carbon monoxide

AbstractIn the past years, whole cell gas toxicity assays have been developed in order to study the harmful effects of gaseous chemicals. Cells are incubated with nutrition medium supplemented with the gaseous substances to be tested. These methods are not feasible for the examination of toxic gases, which are insoluble in water. Carbon monoxide (CO), a poorly soluble and toxic gas (lethal dose 1 vol% for some minutes) has been chosen as a model gas. Besides its binding to hemoglobin, it also leads to an inhibition of cytochrome c oxidase in the mitochondrial respiratory chain of living cells. Thus, a decrease in the respiratory behavior of eukaryotic cells indicates the presence of CO in a gaseous phase. Based on the Bionas® 2500 analyzing system, a device to measure the metabolic and morphological effects, caused by direct gas exposure on eukaryotic cells was established. The gaseous substances directly contact the eukaryotic cell line V79 (Chinese hamster lung fibroblast) without interfering liquid phase in between. Beside the measurement of oxygen consumption also acidification and impedance changes of the cell culture are detected by a metabolic sensor chip. In order to increase the stability different chip surface coatings like collagen A and poly‐L‐lysin (PLL) were used. Adhesion coating leads to a tighter junction of the cells to the sensor chip surface and thus enables higher stability of the confluent cell monolayer towards the gaseous flow during the gas exposure. The method reported here has the potential to become a valuable means for rapid monitoring of toxic compounds in gaseous phases.

Genotoxic assessment on river water using different biological systems

This paper reports genotoxicity and toxicity data in water samples collected in Sinos River, an important water course in the hydrographic region of Guaíba Lake, Rio Grande do Sul State, south of Brazil. This river is exposed to intense anthropic influence by numerous shoes, leather, petrochemical, and metallurgy industries. Water samples were collected at two moments (winter 2006 and spring 2006) at five sites of Sinos River and evaluated using in vitro V79 Chinese hamster lung fibroblasts (cytotoxicity, comet assay and micronucleus test) and Allium cepa test (toxicity and micronucleus test). Comet and micronucleus tests revealed that water samples collected exerted cytotoxic, toxic, genotoxic and mutagenic effects. The results showed the toxic action of organic and inorganic agents found in the water samples in all sites of Sinos River, for both data collections. The main causes behind pollution were the domestic and industrial toxic discharges. The V79 and A. cepa tests were proved efficient to detect toxicity and genotoxicity caused by complex mixtures. This study also showed the need for constant monitoring in sites with strong environmental degradation caused by industrial discharges and urban sewages.

Effect of the dibenzylbutyrolactone lignan (−)-hinokinin on doxorubicin and methyl methanesulfonate clastogenicity in V79 Chinese hamster lung fibroblasts

The dibenzylbutyrolactone lignan (−)-hinokinin (HK) was obtained by partial synthesis from (−)-cubebin, isolated from the dry seeds of the pepper, Piper cubeba. In view of the trypanocidal activity of HK and its potential as a lead compound for drug development, evaluation of its possible genotoxic activity is required. We have tested HK for possible genotoxicity and evaluated the compound's effect on the activity of the clastogens doxorubicin (DXR) and methyl methanesulfonate (MMS) in the micronucleus (MN) assay with Chinese hamster lung fibroblast V79 cells. HK alone did not induce MN, at concentrations up to 128 μM. In combined treatments, HK reduced the frequency of MN induced by MMS. With respect to DXR, HK exerted a protective effect at lower concentrations, but at higher concentrations it potentiated DXR clastogenicity.

Antioxidant activities of major thyme ingredients and lack of (oxidative) DNA damage in V79 Chinese hamster lung fibroblast cells at low levels of carvacrol and thymol

The leafy parts of thyme and its essential oil have been used in foods for the flavour, aroma and preservation and also in folk medicines. In the present study the genotoxicity of thymol and carvacrol was examined using comet assay. In V79 Chinese hamster lung fibroblast cells treated with 1, 5, 25 μM thymol and carvacrol, only 25 μM thymol caused some clastogenic DNA damage. For detection of oxidative DNA damage, the comet assay with formamido pyrimidine glycosylase (Fpg) protein was used: When V79 cells were treated with 1, 5, 25 μM thymol and carvacrol and post-treated with Fpg enzyme, no significant increase of Fpg-sensitive sites was observed at all concentrations studied. Reactive oxygen species (ROS) generation decreased slightly in the presence of thymol (1–100 μM) and carvacrol (5 μM) between 1 and 4 h, yet increased at the highest 100 μM concentration of carvacrol after 24 h. Thymol and carvacrol displayed a concentration dependent antioxidant capacity, whilst γ-terpinene which lacks a phenolic group did not show any antioxidant capacity in the trolox equivalent antioxidant capacity (TEAC) assay. The results of this study indicate a lack of clastogenic activity for thymol and carvacrol at biologically relevant concentrations, and a moderate antioxidant activity in vitro.

SnCl2-induced DNA damage and repair inhibition of MMS-caused lesions in V79 Chinese hamster fibroblasts

In order to clarify the molecular mechanisms of Sn(2+) genotoxicity, we evaluated the induction of strand breaks, formamidopyrimidine DNA glycosylase (Fpg) and endonuclease III (Endo III) sensitive sites, and the interference with the repair of methyl methane sulfonate (MMS)-caused DNA damage in V79 Chinese hamster lung fibroblasts exposed to stannous chloride by comet assay. A concentration-related increase in the DNA damage induced by 2 h SnCl(2) treatment at a concentration range of 50-1,000 microM was observed (r = 0.993; P < 0.01). Significantly elevated DNA migration in relation to the control level was detected at doses 100, 500 and 1,000 microM in normal alkaline and at doses 500 and 1,000 microM in modified (with Fpg and Endo III) comet assay. Although 50 microM SnCl(2) concentration did not increase significantly the DNA migration by itself in comet assay, it was capable to inhibit the repair of MMS-induced DNA damage during the post-treatment period of 24 h. Our results demonstrate the genotoxic and comutagenic effects of stannous chloride in V79 cells. The inhibitory effect of Sn(2+) on repair of MMS-induced DNA damage suggests that this metal can also interfere in DNA repair systems thus contributing to increased mutation by shifting the balance from error-free to error-prone repair processes.

Modulating effects on oxidative DNA damage and antioxidant capacities of thyme and its major ingredients in V79 Chinese hamster lung fibroblast cells

Modulating effects on oxidative DNA damage and antioxidant capacities of thyme and its major ingredients in V79 Chinese hamster lung fibroblast cells

Cytotoxic and genotoxic effects of tambjamine D, an alkaloid isolated from the nudibranch Tambja eliora, on Chinese hamster lung fibroblasts

Marine organisms have been shown to be potential sources of bioactive compounds with pharmaceutical applications. Previous chemical investigation of the nudibranch Tambja eliora led to the isolation of the alkaloid tambjamine D. Tambjamines have been isolated from marine sources and belong to the family of 4-methoxypyrrolic-derived natural products, which display promising immunosuppressive and cytotoxic properties. Their ability to intercalate DNA and their pro-oxidant activity may be related to some of the biological effects of the 4-methoxypyrrolic alkaloids. The aim of the present investigation was to determine the cytotoxic, pro-oxidant and genotoxic properties of tambjamine D in V79 Chinese hamster lung fibroblast cells. Tambjamine D displayed a potent cytotoxic effect in V79 cells (IC50 1.2 μg/mL) evaluated by the MTT assay. Based on the MTT result, V79 cells were treated with different concentrations of tambjamine D (0.6, 1.2, 2.4 and 4.8 μg/mL). After 24 h, tambjamine D reduced the number of viable cells in a concentration-dependent way at all concentrations tested, assessed by the trypan blue dye exclusion test. The hemolytic assay showed that the cytotoxic activity of tambjamine D was not related to membrane disruption (EC50 > 100 μg/mL). Tambjamine D increased the number of apoptotic cells in a concentration-dependent manner at all concentrations tested according to acridine orange/ethidium bromide staining, showing that the alkaloid cytotoxic effect was related to the induction of apoptosis. MTT reduction was stimulated by tambjamine D, which may indicate the generation of reactive oxygen species. Accordingly, treatment of cells with tambjamine D increased nitrite/nitrate at all concentrations and TBARS production starting at the concentration corresponding to the IC50. Tambjamine D, also, induced DNA strand breaks and increased the micronucleus cell frequency as evaluated by comet and micronucleus tests, respectively, at all concentrations evaluated, showing a genotoxic risk induced by tambjamine D.

Investigating the Temporal Effects of Respiratory-Gated and Intensity-Modulated Radiotherapy Treatment Delivery on In Vitro Survival: An Experimental and Theoretical Study

To experimentally and theoretically investigate the temporal effects of respiratory-gated and intensity-modulated radiotherapy (IMRT) treatment delivery on in vitro survival. Experiments were designed to isolate the effects of periodic irradiation (gating), partial tumor irradiation (IMRT), and extended treatment time (gating and IMRT). V79 Chinese hamster lung fibroblast cells were irradiated to 2 Gy with four delivery methods and a clonogenic assay performed. Theoretical incomplete repair model calculations were performed using the incomplete repair model. Treatment times ranged from 1.67 min (conformal radiotherapy, CRT) to 15 min (gated IMRT). Survival fraction calculations ranged from 68.2% for CRT to 68.7% for gated IMRT. For the same treatment time (5 min), gated delivery alone and IMRT delivery alone both had a calculated survival fraction of 68.3%. The experimental values ranged from 65.7% +/- 1.0% to 67.3% +/- 1.3%, indicating no significant difference between the experimental observations and theoretical calculations. The theoretical results predicted that of the three temporal effects of radiation delivery caused by gating and IMRT, extended treatment time was the dominant effect. Care should be taken clinically to ensure that the use of gated IMRT does not significantly increase treatment times, by evaluating appropriate respiratory gating duty cycles and IMRT delivery complexity.

Genotoxicity of aminohydroxynaphthoquinones in bacteria, yeast, and Chinese hamster lung fibroblast cells

There are few studies on the biological activity of aminohydroxy derivates of 1,4-naphthoquinone (1,4-NQ) on prokaryotic and eukaryotic cells. We determined the mutagenic activity of 5-amino-8-hydroxy-1,4-naphthoquinone (ANQ) and 5-amino-2,8-dihydroxy-1,4-naphthoquinone (ANQ-OH) as compared to the unsubstituted 1,4-NQ in Salmonella/microsome assay. Potential mutagenic and recombinogenic effects and cytotoxicity were analyzed in haploid and diploid cultures of the yeast Saccharomyces cerevisiae. In Salmonella/microsome assay, 1,4-NQ was not mutagenic, whereas aminohydroxynaphthoquinones were weakly mutagenic in TA98 and TA102 strains. In haploid yeast in stationary growth phase (STAT), mutagenic response was only observed for the hom3 locus at the highest dose. In diploid yeast, aminohydroxynaphthoquinones did not induce any recombinogenic events, but 1,4-NQ was shown to be a recombinogenic agent. These results suggest that aminohydroxynaphthoquinones are weak mutagenic agents only in prokaryotic cells. The cytotoxicity of 1,4-NQ in yeast stationary cells was more significant in diploid cells as compared to that observed in haploid cells. However, ANQ and ANQOH were slightly cytotoxic in all treatments. Genotoxicity of these naphthoquinone compounds was also determined in V79 Chinese hamster lung fibroblast cells using standard Comet, as well as modified Comet assay with the bacterial enzymes formamidopyrimidine DNA-glycosylase (FPG) and endonuclease III (ENDOIII). Both 1,4-NQ and ANQ induced pronounced DNA damage in the standard Comet assay. The genotoxic effect of ANQ-OH was observed only at the highest dose. In presence of metabolic activation all substances showed genotoxic effects on V79 cells. Post-treatment of V79 cells with ENDOIII and FPG proteins did not have a significant effect on ANQ-OH-induced oxidative DNA damage as compared to standard alkaline Comet assay. However, all naphthoquinones were genotoxic in V79 cells in the presence of metabolic activation and post-treatment with enzymes, indicating that all compounds induced oxidative DNA damage in V79 cells. Our data suggest that aminohydroxynaphthoquinone pro-oxidant activity, together with their capability of DNA intercalation, have an important role in mutagenic and genotoxic activities.

Toxicity of Parked Motor Vehicle Indoor Air

The interior of motor vehicles is made of a wide variety of synthetic materials, which emit volatile organic compounds (VOC). We tested the health effects of emissions from vehicles exposed to "parked in sunshine" conditions. A new and a 3 year old vehicle with identical interior were exposed to 14 000 W of light. Indoor air was analyzed by GC-MS. Toxicity of extracts of indoor air was assayed in human primary keratinocytes, human lung epithelial A549 cell line, and Chinese hamster V79 lung fibroblasts. In addition, toxicity after metabolic activation by CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, and CYP2E1 was assayed. The effect on type I allergic reaction (IgE-mediated immune response), type IV allergic reaction (T-cell mediated immune response), and irritative potential was evaluated also. A total of 10.9 and 1.2 mg/m(3) VOC were found in new and used motor vehicle indoor air, respectively. The major compounds in the new vehicle were o,m,p-xylenes, C3 and C4-alkylbenzenes, dodecane, tridecane, and methylpyrrolidinone. In the used vehicle they were acetone, methylpyrrolidinone, methylcyclohexane, acetaldehyde, o,m,p-xylenes, ethylhexanol, and toluene. No toxicity was observed in any cell line with or without metabolic activation. Neither did we find an effect on type IV sensitization or an irritative potential. A slight but statistically significant aggravating effect on IgE-mediated immune response of only the new vehicle indoor air was determined (p < 0.05). The IgE-response modulating effect of indoor air might be relevant for atopic individuals. Else no direct toxicity, no toxicity after metabolic activation by cytochrome P450, and no irritative or type IV sensitizing potential of motor vehicle indoor air were found, neither from the new nor used vehicle. Our investigations indicated no apparent health hazard of parked motor vehicle indoor air.

Interaction of piroxicam with mitochondrial membrane and cytochrome c

Modulation of surface properties of biomembranes by any ligand leading to permeabilization, fusion, rupture, etc. is a fundamental requirement for many biological processes. In this work, we present the interaction of piroxicam, a long acting Non-Steroidal Anti-Inflammatory Drug (NSAID) with isolated mitochondria, membrane mimetic systems, intact cells and a mitochondrial protein cytochrome c. Dye permeabilization study on isolated mitochondria indicates that piroxicam can permeabilize mitochondrial membrane. Direct imaging by Scanning Electron Microscope (SEM) shows that piroxicam induces changes in mitochondrial membrane morphology leading to fusion and rupture. Transmission Electron Microscope (TEM) imaging of piroxicam treated DMPC vesicles and mixed micelles formed from CTAB and SDS show that causing membrane fusion is a general property of piroxicam at physiological pH. In intact cells viz., V79 Chinese Hamster lung fibroblast, piroxicam is capable of releasing cytochrome c from mitochondria into the cytosol in a dose dependent manner along with the enhancement of downstream proapoptotic event viz., increase in caspase-3 activity. We have also shown that piroxicam can reduce cytochrome c within a time frame relevant to its lifetime in blood plasma. UV-visible spectroscopy has been used to study the reaction mechanism and kinetics in detail, allowing us to propose and validate a Michaelis–Menten like reaction scheme. CD spectroscopy shows that small but significant changes occur in the structure of cytochrome c when reduced by piroxicam.

CYP2D6 increases toxicity of the designer drug 4-methylthioamphetamine (4-MTA)

4-Methylthioamphetamine (4-MTA) belongs to a group of new amphetamine derivatives that is usually sold as “ecstasy” or “flatliners” on the illicit drug market. Large interindividual differences in 4-MTA mediated toxicity have been reported in humans. Therefore, we tested whether CYP2D6 or its variant alleles as well as CYP3A4 influence the susceptibility to 4-MTA. For this purpose, we used the colony formation assay with Chinese hamster lung fibroblast V79 cells expressing human wild-type CYP2D6 (CYP2D6*1), the low activity alleles CYP2D6*2, CYP2D6*9, as well as human CYP3A4. The obtained results showed that the expression of wild type CYP2D6*1 clearly enhanced the susceptibility to the cytotoxic effects of 4-MTA compared with the parental cells devoid of CYP-dependent enzymatic activity. Toxicity in V79 CYP2D6*1 was also higher compared to the V79 cell lines expressing the low activity alleles CYP2D6*2 and CYP2D6*9. In contrast to CYP2D6, the CYP3A4 isoenzyme did not enhance 4-MTA toxicity. In conclusion, our results suggest that CYP2D6 rapid metabolizers may be more susceptible to 4-MTA toxicity than CYP2D6 poor metabolizers.

In vitro cytotoxic activities of (+)-usnic acid and (−)-usnic acid on V79, A549, and human lymphocyte cells and their non-genotoxicity on human lymphocytes

This study investigates cytotoxic and genotoxic activities of (+)-Usnic acid and (−)-usnic acid isolated from the lichen Ramalina farinacea and the lichen Cladonia foliacea, respectively. To determine the activities of these acids, we used the MTT assay on V79 (Chinese hamster lung fibroblast like) and A549 (human lung carcinoma epithelial like) cell lines and cytokinesis-blocked micronucleus (CBMN) assay in human lymphocytes in vitro. Our results suggest that both enantiomers of usnic acid are non-genotoxic shown by the absence of micronucleus induction in human lymphocytes and have significant cytotoxic and apoptotic effects to induce cell killing in cultured human lymphocytes, V79 and A549 cell lines. Even low doses of (+)-usnic acid showed high cytotoxic activity against cancerous cells. The MTT results and cell proliferation index (CPI) values based on the CBMN test results are found in good agreement.