Inhibition of glycoconjugate secretion by colchicine and cytochalasin B

Summary The effects have been analyzed of cytochalasin B and colchicine on the secretion of glycoconjugates by human bronchial expiants labeled in vitro with radioactive glucosamine. Both cytochalasin B and colchicine had no effect on baseline ¹⁴C-labeled glycoconjugate release but caused a dose-dependent (10^–7–10^–4 M) inhibition of ¹⁴C-glycoconjugate release and discharge of labeled macromolecules from mucous and serous cells induced by 5 · 10^–5 M methacholine.Quantitative autoradiographic analyses showed that neither cytochalasin B nor colchicine inhibited ³H-threonine or ³H-glucosamine incorporation into mucous and serous cells of the submucosal glands or goblet cells of the airway epithelium. Colchicine (10^–5 M) but not cytochalasin B significantly reduced the rate at which labeled macromolecules were transported through mucous, serous and goblet cells but this effect was not observed until 4 h after the addition of colchicine. Neither cytochalasin B nor colchicine affected the basal rate of labeled-macromolecule discharge from mucous, serous or goblet cells. At a concentration of 10^–5 M, both agents completely inhibited the increase in labeled-macromolecule discharge induced in mucous and serous cells by methacholine.Our results suggest that in the submucosal gland of human airways microtubules and microfilaments may be important in secretagogue-induced but not in baseline cellular glycoconjugate discharge, implying that the mechanisms of the two processes differ significantly. Furthermore, a role for microtubules is suggested in the transport of secretory granules through mucous, serous and goblet cells.Supported by National Institutes of Health Research Grant 5R01HL22444. The authors gratefully acknowledge the technical assistance of Mr. Tudor Williams, Mr. Eduardo Quintanilla and Ms. Maureen Hayes     

Metal requirement of the isolated red cell Ca-pump ATPase after elimination of calmodulin dependence by trypsin attack

Mild proteolysis by trypsin activates the purified (Ca²⁺ + Mg²⁺) - ATPase protein from human red cells in a way which is similar to the effect obtained by addition of calmodulin. The trypsin concentration required to reach half maximal effect in 3 minutes at 37°C is 2.5 – 3.5 μg/ml. SDS-poly-acrylamide gel electrophoresis reveals a degradation of the main protein (150'000 Dalton) into a large fragment (95'000 – 100'000 Dalton) and a small fragment (35'000 – 40'000 Dalton). Increasing ATPase activity correlates with the degree of proteolysis.The _Ca of the digested (Ca²⁺ + Mg²⁺)-ATPase is 0.85 ± 0.1 μM Ca²⁺ as compared to 8.0 ± 0.75 μM Ca²⁺ before digestion and is statistically significantly different from _Ca = 1.66 ± 0.22 μM Ca²⁺ observed in activation by a saturating calmodulin concentration. Addition of calmodulin to the trypsinized enzyme has neither an effect on the Ca²⁺-affinity nor achieves any large increase of the maximal rate.High Ca²⁺ concentrations (above 0.05 – 0.1 mM) after trypsin treatment still inhibit the (Ca²⁺ + Mg²⁺)-ATPase activity. Mg²⁺ activates in the same concentration range ( _Mg = 25 μM) as in the undigested preparation ( _Mg = 27 μM) and retains its competitive behaviour towards Ca²⁺ after trypsin treatment.It is concluded that (1) trypsin treatment unmasks high affinity sites for Ca²⁺ ( _Ca 1 μM) and that, therefore, such sites are not added to the system by calmodulin, and (2) that inhibition by high Ca²⁺-concentrations is not due to Ca - Mg competition at sites located on the calmodulin molecule.     

Regulation of phospholipase D activity in synaptosomes permeabilized with Staphylococcus aureus α-toxin

In order to investigate the regulation of presynaptic phospholipase D (PLD) activity by calcium and G proteins, we established a permeabilization procedure for rat cortical synaptosomes using Staphylococcus aureus α-toxin (30–100 μg/ml). In permeabilized synaptosomes, PLD activity was significantly stimulated when the concentration of free calcium was increased from 0.1 μM to 1 μM. This activation was inhibited in the presence of KN-62 (1 μM), an inhibitor of calcium/calmodulin-dependent kinase II (CaMKII), but not by the protein kinase C inhibitor, Ro 31-8220 (1–10 μM). Synaptosomal PLD activity was also stimulated in the presence of 1 μM GTPγS. When Rho proteins were inhibited by pretreatment of the synaptosomes with Clostridium difficile toxin B (TcdB; 1–10 ng/ml), the effect of GTPγS was significantly reduced; in contrast, brefeldin A (10–100 μM), an inhibitor of ARF activation, was ineffective. Calcium stimulation of PLD was inhibited by TcdB, but GTPγS-dependent activation was insensitive to KN-62. We conclude that synaptosomal PLD is activated in a pathway which sequentially involves CaMKII and Rho proteins.     

Ascorbic acid promotes prostanoid release in human lung parenchyma

Ascorbic acid reduces airway reactivity to inhaled bronchoconstrictor agents in man and guinea pigs. The precise mechanism(s) responsible for this effect are unknown, but in both species an acute indomethacin treatment reverses the action of the ascorbic acid. To determine if ascorbic acid promotes prostanoid synthesis and/or inhibits degradation, human lung parenchymal slices (100–200mg) were incubated for 60 minutes in oxygenated Tyrode's solution alone or with sodium ascorbate (0.001M–1M) and/or methacholine (1μM–100μM) and/or indomethacin (0.17μM–17μM). Aliquots of the incubation medium were assayed by radioimmunoassay for PGE₂, PGF_2α, thromboxane B₂ and 6-keto-PGF_1α. Ascorbic acid increased the accumulation of all four prostanoids in the incubation medium, especially thromboxane B₂ and 6-keto-PGF_1α. This stimulatory effect of ascorbic acid was concentration-dependent and was inhibited by indomethacin. We conclude that ascorbic acid can alter prostanoid generation by human lung tissue and this effect may, in part, explain its antibronchoconstrictor activity in man.     

Inhibitory effects of a lipoxygenase inhibitor nordihydroguaiaretic acid on antagonism of leukotriene C4-induced contractions of isolated guinea-pig trachea

We have studied the effects of a lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) on antagonim of leukotriene (LT) C₄-induced contractions of isolated guinea-pig trachea and the results were compared to that of a cycloocygenase inhibitor indomethacin, NDGA (30 μM) as well as idomethacin (5 μM) inhibited LTC₄-iduced contraction. But in the presence of indomethacin NDGA was ineffective to inhibit the LTC₄ response, whereas two other lipoxygenase inhibitors, phenidone (3–30 μM) and 5,8,11,14-eicostatetraynoic acid (ETYA, 10 μM), markedly inhibited it. The antagonist action of an LTD₄ receptor antagonist FPL55712 against LTC₄-induced contractions was significantly reduced by NDGA (10–30 μM), but indomethacin had no effect on it. NDGA possessed the same inhibitory effect n the LTC₄ antagonism in the presence of indomethacin, but 0.3 μM phenidone and 1 μM ETYA which did not inhibit the LTC₄ response had no effect on it. NDGA also inhibited the relaxant response of isoproterenol on the contraction elicited by 30 nM LTC₄, but did not affect those of forskolin and aminophylline. The relaxant response of isoproterenol on the LCT₄ response was not inhibited by indomethacin, 0.3 μM phenidone and 1 μM ETYA. In the presence of a γ-glutamyltranspeptidase inhibitor, L-serine borate (SB, 45 mM), NDGA had no effect on the LTC₄ antagonism and the relaxant response of isoproterenol. In contrast, NDGA significantly inhibited the relaxant response of isoproterenol on 30 μM histamine- and 30 μM acetylcholine-induced contractions, but it did not affect the histamine antagonism by a histamine H₁-blocker pyrilamine. These results suggest that some putative nonprostanoids are involved in LTC₄-induced contractions of guinea-pig trachea and which regulate the effects of LTD₄ antagonism and β-adrenoceptor activation.     

Inactivation by chloroquine of α-galactosidase in cultured human skin fibroblasts

When human skin fibroblasts are cultured in the presence of chloroquine or NH₄Cl there is a decrease in the intracellular level of lysosomal hydrolases and a concomitant increase in the extracellular activity as compared with cells grown in the absence of a base (cf [18]). In a medium with 25 μM chloroquine or 5 mM NH₄Cl, the decrease in the intracellular activity of β-hexosaminidase, arylsulphatase and β-glucuronidase is 10–40% after 1 day. A similar decrease in α-galactosidase activity is observed in cells grown in the presence of 5 mM NH₄Cl. However, in the presence of 25 μM chloroquine, the intracellular activity of α-galactosidase decreases by 80–90% within 6 h. The inactivation is irreversible. After removal of the chloroquine and further culture of the cells in chloroquine-free medium, α-galactosidase activity gradually increases due to de novo synthesis. The turnover time of α-galactosidase was calculated to be 1.9 days. Inactivation of α-galactosidase also occurs when homogenates are incubated with chloroquine, but the concentration of the base required for maximum inactivation is at least three orders of magnitude higher than that which must be present in the medium of intact cells to obtain the same effect.     

EFFECT OF SUBLETHAL DOSES OF CADMIUM, INORGANIC MERCURY AND METHYLMERCURY ON THE CELL MORPHOLOGY OF AN INSECT CELL LINE (AEDES ALBOPICTUS, C6/36)

The effect of CdCl₂(44 μ ), HgCl₂(3.7 μ ), and MeHgCl (2 μ ) on the morphology ofAedes albopictusC6/36 cells was studied at the light microscopical level. Treatment times and metal concentrations were in the sublethal range as determined by a fluorometric dye exclusion test. The three metal species had profound effects on the cell morphology. MeHgCl treatment induced the development of a large number of short, actin-supported, tangled filopodia. Both CdCl₂and HgCl₂induced long extensions. Pretreatment with colchicine but not with cyto-chalasin B prevented formation of these extensions which suggests that they were supported by microtubules. This was confirmed by immunostaining for microtubules. The extensions were relatively stable towards colchicine post-treatment. To authors’ knowledge, this effect has not yet been described for heavy metals. The similarity with 20-hydroxyecdysone-treated cells and the occurrence of cytoplasmic feet in insect cells is discussed.     

Effect of Rho-associated kinase (ROCK) inhibitor Y-27632 on the post-thaw viability of cryopreserved human bone marrow-derived mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (MSC) have previously been reported to be susceptible to cryopreservation-induced apoptosis. A significant fraction of MSC lose their viability during freeze-thawing, which represent a major technical barrier in attaining adequate viable cell numbers for optimal efficacy in transplantation therapy. Recently, it was reported that a Rho-associated kinase (ROCK) inhibitor Y-27632 could enhance the post-thaw viability and physiological function of cryopreserved human embryonic stem cells (hESC). Hence, this study attempted to investigate whether Y-27632 can exert a similar beneficial effect on the post-thaw viability of cryopreserved MSC. A concentration range of 1–100 μM Y-27632 was supplemented in both the cryopreservation medium (10% (v/v) dimethyl sulfoxide), as well as the post-thaw culture medium. The supplementation of Y-27632 had no significant effect on the immediate post-thaw viability, as assessed by trypan blue exclusion. However, 24 h after the frozen-thawed cell suspensions were re-plated on new cell culture dishes (with varying concentrations of Y-27632 within the post-thaw culture media); the MTT assay subsequently showed significant differences in the proportion of adherent viable cells over the concentration range of Y-27632 examined, with a peak at between 5 and 10 μM. At zero concentration of Y-27632, the proportion of viable adherent cells was 39.8 ± 0.9%; and this value peaked at 48.5 ± 1.7% with 5 μM Y-27632 and 48.4 ± 1.8% with 10 μM Y-27632, prior to decreasing to 36.0 ± 0.6% with 100 μM Y-27632. Additionally, it was observed that Y-27632 induced morphological changes in the frozen-thawed MSC. With increasing Y-27632 concentration, the cells displayed more extensive branching of cytoplasmic extensions that gave a ‘web-like’ appearance. This is consistent with previous reports of Y-27632 stimulating neuronal differentiation of MSC.     

A radioligand binding assay for antitubulin activity in tumor cells

The benzamide RH-5854 is shown to be highly potent toward tumor cells and to arrest nuclear division by a highly specific covalent binding to the beta-subunit of tubulin in the colchicine binding region. Binding of 3H-RH-5854 to beta-tubulin in HCT-116 colon cancer cells is saturable and has been exploited in the development of a cell-based competitive binding assay, which allows antitubulin effects to be detected in whole cells. 3H-RH-5854 binding is strongly inhibited by preincubating the cells with compounds that bind to the colchicine site and with paclitaxel. Binding of 3H-RH-5854 is enhanced by preincubating the cells with vinblastine but not by other agents that bind at or near the vinblastine site (ansamitocin P-3 and phomopsin A). Various cytotoxic agents that do not act on tubulin do not affect binding of 3H-RH-5854 in HCT-116 cells, demonstrating specificity of the assay for detection of antitubulin activity. As an alternative to traditional assays that employ isolated brain tubulin, the 3HRH-5854 binding assay enables screening for antitubulin effects directly in tumor cells, providing an assay that accounts for cell-specific criteria that influence sensitivity such as different tubulin isotypes, tubulin mutations, drug metabolism, and efflux mechanisms.     

Protective effect of quercetin and luteolin in human melanoma HMB-2 cells

Multifunctional effects of flavonoids are reported to be markedly connected with their structure and the functional groups in the molecule. The important role in the activity play C2–C3 double bond, hydroxyl group at C3 and the number of hydroxyl groups at phenyl ring (B). In this paper, the DNA protective free radical scavenging potential of quercetin (QU) and luteolin (LU) against H₂O₂ and their clastogenic effect alone and in combination with melphalan (MH) were investigated in human melanoma HMB-2 cells. Elevated frequency of chromosomal aberrations induced by MH, that at high doses have shown a variety of toxic side effects, was statistically decreased by studied flavonoids regarding to control (QU at the concentration of 50 μM and LU already at the concentration of 20 μM). The results concerning DNA protective potential against free radicals in HMB-2 cells demonstrated that QU and LU have significant effect in dose dependent manner. The percentage of QU protective effect is 40% at the concentration 20 μM, resp. 80% at the concentration 100 μM. Comparable values were obtained with LU. Results are correlated to their structural arrangement and organization of the hydroxyl groups.     

Oxidative burst inhibitory and cytotoxic amides and lignans from the stem bark of Fagara heitzii (Rutaceae)

Two amides, heitziamide A and heitziamide B and two phenylethanoids, heitziethanoid A and heitziethanoid B together with thirteen known compounds were isolated from F. heitzii (Letouzey). The structures of all compounds were established by spectroscopic analysis.Nine compounds were evaluated for oxidative burst inhibitory activity in a chemoluminescence assay and for cytotoxicity against PC-3 prostate cancer cells.All compounds exhibited a clear suppressive effect on phagocytosis response upon activation with serum opsonized zymosan at the range of IC₅₀ = 2.0–6.5 μM, but no cytotoxic effect was observed (IC₅₀ > 100 μM).     

The influence of xanthines on the contractile responses of PGF2α and PGD2 in human parenchymal strips

Prostaglandins (PGs) F_2α and D₂ are bronchoconstrictor agents which are released under allergic conditions such as asthma. The efficacy and potency of PGF_2α and PGD₂ differ in some tissues. We compared the effects of these two PGs in sensitized human parenchymal strips. In six experiments, PGF_2α 0.1 and 0.3 μM produced greater contractions than PGD₂ at the same concentrations. There were no significant differences between the contractions from the two PGs at concentrations of 0.01, 0.03, 1.0–10 μM and the two PGs appeared to be equipotent.We studied the effects of the anti-asthmatic drug theophylline, and its analogue enprofylline, on the contraction caused by these PGs. Theophylline 100 μM caused no change to the cumulative concentration response curves. However, enprofylline 100 μM reduced the PGF_2α-induced contractions.     

Effect of calcium(ion) uptake by rat adrenal mitochondrial on pregnenolone formation and spectral properties of cytochrome P-450

The effect of calcium on pregnenolone formation from endogenous precursors has been studied in mitochondria from rat decapsulated and capsular adrenalgglands. In the presence of succinate, addition of calcium chloride in the concentration range 20–150 μM caused an inhibition of pregnenolone formation of added deoxycosticosterone in decapsulated adrenal mitochondria was also inhibited. Under these conditions, calcium inhibited the reduction of adrenodoxin, a component of the cytochrome P-450 reductase system, presumably because uptake of calcium by the mitochondria competes with energy-linked transhydrogenase for high-energy intermediates. For this reason, incubations were carried out in the presence of succinate plus isocitrate plus NADP⁺. Under these conditions, calcium chloride in the concentration range 120–875 μM caused a 2–4-fold stimulation of pregnenolone formation, but had no effect on corticosterone formation from added deoxycorticosterone.The effect of calcium on the optical spectra of cytochrome P-450 has also been examined in mitochondria from decapsulated and capsular rat adrenals. In the presence of succinate, calcium induced a spectral change resembling a type I difference spectrum of cytochrome P-450. Thus it appears that uptake of calcium the interaction of mitochondrial cytochrome P-450 with endogenous substrate.     

Effects of four agents that modify microtubules and microfilaments (vinblastine,colchicine, lidocaine,and cytochalasin B) on macrophage-mediated cytotoxicity to tumor cells

Summary The effects of vinblastine, colchicine, lidocaine, and cytochalasin B on tumor cell killing by BCG-activated macrophages were examined. These four drugs were selected for their action on membrane-associated cytoskeletal components, microtubules, and microfilaments. Colchicine and vinblastine, which block microtubular synthesis, inhibit macrophage-mediated tumor-cell cytotoxicity at a concentration of 10^–6 M. Cytochalasin B, which disrupts microfilaments, enhances tumor cell lysis and stasis due to activated macrophages at a concentration of 10^–7 M. Lidocaine, which may induce the disappearance of both microtubules and microfilaments, has the same inhibiting effect as vinblastine at a concentration of 5×10^–7 M. Whereas vinblastine and lidocaine seem to act on the macrophage itself, cytochalasin B exerts its effect predominantly on the tumor cell. These results suggest that microtubules and microfilaments play a role in the destruction of tumor cells by activated macrophages.     

Acrylamide and glycidamide: genotoxic effects in V79-cells and human blood

Acrylamide (AA) can be formed in certain foods by heating, predominantly from the precursor asparagine. It is a carcinogen in animal experiments, but the relevance of dietary exposure for humans is still under debate. There is substantial evidence that glycidamide (GA), metabolically formed from AA by Cyp 2E1-mediated epoxidation, acts as ultimate mutagenic agent. We compared the mutagenic potential of AA and GA in V79-cells, using the hprt mutagenicity-test with N-methyl-N′-nitro-N-nitroso-guanidine (MNNG) as positive control. Whereas MNNG showed marked mutagenic effectivity already at 0.5 μM, AA was inactive up to a concentration of 10 mM. In contrast, GA showed a concentration dependent induction of mutations at concentrations of 800 μM and higher. Human blood was used as model system to investigate genotoxic potential in lymphocytes by single cell gel electrophoresis (comet assay) and by measuring the induction of micronuclei (MN) with bleomycin (BL) as positive control. AA did not induce significant genotoxicity or mutagenicity up to 6000 μM. With GA, concentration dependent DNA damage was observed in the dose range of 300–3000 μM after 4 h incubation. Significant MN-induction was not observed with AA (up to 5000 μM) and GA (up to 1000 μM), whereas BL (4 μM) induced significantly enhanced MN frequencies. Thus, in our systems GA appears to exert a rather moderate genotoxic activity.     

The influence of contact-inhibited growth and of agents which alter cell morphology on the levels of G- and F-actin in cultured cells

The amounts of G-actin and F-actin were measured in cultured cells grown under various conditions. The percent of total actin as F-actin in monolayer cultures of asynchronous cells was 72.4% in Chinese hamster ovary (CHO) cells, 57.7% in HeLa cells, 69.8% in V79 cells, and 79.5% in 1080 cells. Actin comprises 2.4–3.1% of the total protein in these cell lines. Treatment of cells with 20 μM cytochalasin B (CB) caused different cytological effects from treatment with 10 μM colchicine, but the effects characteristic of each drug were observed throughout the range of cell lines used. Of the five cell lines treated with CB only the V79 and CHO cells showed a decrease (5–8%) in the level of F-actin. Colchicine treatment of HeLa cells resulted in a 13% increase in the percent F-actin, but similar treatment of CHO cells caused no significant change in F-actin. Therefore, a change in the steady state level of F-actin is not necessary for the observed cell shape change. The F-actin levels in CHO cells treated with 7 mM procaine decreased from 72 to 65% over the first 15 min of exposure, a time during which the cells rounded. After continuous exposure of the cells to procaine for 1 h, the F-actin percentage returned to control levels and the cells, though abnormal in appearance, flattened on the culture dish. The relationship between the level of F-actin and cell density was studied in the 10T_1/2 cells, a cell line which demonstrates density-dependent growth regulation. While contact inhibition was accompanied by a decrease in the F-actin percentage (from >95% to about 60%) in one strain of 10T_1/2 cells, two other strains of the same cells progressed from log phase growth (highly motile) to late plateau phase (non-motile, contact-inhibited) with a constant level of F-actin (about 60%). A spontaneous transformant of this cell line, which no longer demonstrated contact-inhibited cell growth, also maintained the same constant F-actin (60%). Thus, the maintenance of contact-inhibited growth control does not appear to depend upon the net distribution of actin between the globular and filamentous forms.     

Monitoring of α-ketoglutarate in a fermentation process using expanded bed enzyme reactors

A bienzyme flow injection system is presented for the monitoring of α-ketoglutarate produced in a fermentation process, using glutamate dehydrogenase (GDH) and glutamate oxidase (GlOx) immobilised in two serially connected expanded bed reactors. The use of expanded bed resulted in unhindered passage of the bacterial cells through the columns, and thereby the need of a separate filtering step (e.g. microdialysis) was avoided. In the first reactor, α-ketoglutarate was converted to -glutamate by GDH in the presence of ammonia and NADH. In the following reactor, -glutamate was converted by GlOx to α-ketoglutarate, ammonia and hydrogen peroxide, which was detected in an electrochemical flow-through cell at +650 mV vs. Pt/(0.1 M KCl). The detection limit of α-ketoglutarate in the coupled packed bed reactors was 1 μM (defined as 3 S/N), the linear range 0–100 μM, and the sensitivity 0.80 nA/μM (R² 0.99). In the coupled expanded bed reactors, the detection limit of α-ketoglutarate was 7 μM (defined as 3 S/N), the linear range and the sensitivity being 0–500 μM and 0.11 nA/μM (R² 1.00), respectively. The response time (defined as the time between peak rise and return to baseline) was 5 min for coupled packed beds (injection of supernatant), and 12 min for coupled expanded beds (injection of sample containing cellular and particulate matter). Several other parameters, such as reactor stability, flow rate dependency, bed expansion, glutamate interference, etc. were investigated and characterised. When analysing real samples from a fermentation broth, the same results were obtained independent of the nature of the reactor system (packed or expanded bed). The hereby described system can easily be automatised and controlled from a personal computer.     

Determination of nelfinavir, a potent HIV protease inhibitor, and its active metabolite M8 in human plasma by high-performance liquid chromatography with photodiode-array detection

We developed and characterized a high-performance liquid chromatographic assay for the determination of nelfinavir (NFV), a potent HIV protease inhibitor, and its active metabolite M8 in human plasma. Extraction of the internal standard, M8 and NFV from the plasma buffered at pH 9.5 was achieved by a liquid–liquid extraction with a mixture of methyl-tert.-butyl ether and hexane. Following two washes of the reconstituted sample with hexane, separation was achieved on an octadecylsilyl analytical column with a mobile phase containing 0.1% trifluoroacetic acid–acetonitrile–methanol (51:46:5, v/v). Detection was performed using an ultraviolet photodiode-array detector. The signal was monitored at a wavelength of 220 nm. The assay was found to be linear and has been validated over the concentration range of 25 to 3000 μg/l for M8 and 25 to 6000 μg/l for NFV, from 500 μl of plasma. Recoveries were 98.9% (SD 8.9%), and 100.2% (SD 11.7%) for M8 and NFV, respectively. Concentrations that gave a signal-to-noise ratio of three (15 μg/l for both M8 and NFV) were selected to determine the limit of detection. The lower limit of quantification (25 μg/l for both M8 and NFV) was defined as the concentration for which the relative standard deviation and the percent deviation from the nominal concentration were lower than 20%.     

Genotoxic effect and nitrative DNA damage in HepG2 cells exposed to aristolochic acid

Aristolochic acid (AA), extensively used as a traditional herbal medicine, was withdrawn from the market in the last century because it was found to be a potent carcinogen in humans and animals. The aim of this study was to evaluate the genotoxic effect of AA and obtain further insight into whether the nitrative DNA damage can be induced by reactive nitrogen species (RNS), including nitric oxide (NO) and its derivative peroxynitrite (ONOO⁻) using human hepatoma HepG2 cells. To identify the genotoxic effect, the comet assay and micronucleus test (MNT) were performed. In the comet assay, 25–200 μM of AA caused a significant increase of DNA migration in a dose-dependent manner. A significant increase of the frequency of micronuclei was found in the range between 12.5 and 50 μM in the MNT. The results showed that AA caused DNA and chromosome damages. To elucidate the nitrative DNA damage mechanism, the level of nitrite and 8-hydroxydeoxyguanosine (8-OHdG), which can be generated by ONOO⁻, were monitored with the 2,3-diaminonaphthalene (DAN) assay and immunoperoxidase staining, respectively. The results showed that AA causes a significant increase in the levels of NO and formation of 8-OHdG at concentrations ≥50 μM. This observation supports the assumption that AA could exert genotoxicity probably via NO and its derivatives at higher concentrations in HepG2 cells.     

Tests for genotoxicity and mutagenicity of furan and its metabolite cis-2-butene-1,4-dial in L5178Y tk mouse lymphoma cells

Furan is found in various food items and is cytotoxic and carcinogenic in the liver of rats and mice. Metabolism of furan includes the formation of an unsaturated dialdehyde, cis-2-butene-1,4-dial (BDA). In view of the multifunctional electrophilic reactivity of BDA, adduct formation with protein and DNA may explain some of the toxic effects. Short-term tests for genotoxicity of furan in mammalian cells are inconclusive, little is known for BDA. We investigated BDA generated by hydrolysis of 2,5-diacetoxy-2,5-dihydrofuran for genotoxicity in L5178Y tk^+/− mouse lymphoma cells using standard procedures for the comet assay, the micronucleus test, and the mouse lymphoma thymidine kinase gene mutation assay, using 4-h incubation periods. Cytotoxicity was remarkable: cell viability at concentrations ≥50 μM was reduced to <50%. In the dose range up to 25 μM, viability was >90%. Measures of comet-tail length and thymidine–kinase mutant frequency were increased 1.6- and 2.4-fold above control, respectively. Analysis of three fully independent replicates with a linear mixed-effects model showed a highly significant increase with concentration for both endpoints. Compared to methyl methanesulfonate used as a positive control, BDA was of similar potency with respect to genotoxicity, but it was much more cytotoxic. Furan added to cell cultures at doses that resulted in time-averaged effective concentrations of up to 3100 μM was neither cytotoxic nor genotoxic. A potential cross-linking activity of BDA was investigated by checking whether gamma radiation-induced DNA migration in the comet assay could be reduced by pre-treatment with BDA. In contrast to the effect of the positive control glutaraldehyde, BDA treatment did not reduce the comet tail length. On the contrary, an increase was observed at ≥100 μM BDA, which was attributable to early apoptotic cells. Although BDA was found to be a relatively potent genotoxic agent in terms of the concentration necessary to double the background measures, cytotoxicity strongly limited the concentration range that produced interpretable results. This may explain some of the inconclusive results and indicates that non-genotoxic effects must be taken into account in the discussion of the modes of toxic and carcinogenic action of furan.