Toxicology in Vitro

Published by Elsevier
Online ISSN: 0887-2333
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Article
At its 25th meeting the ECVAM Scientific Advisory Committee (ESAC) unanimously endorsed that the SkinEthic Reconstructed Human Epidermis (RHE) model could be used for distinguishing between corrosive and non-corrosive chemicals within the context of the Organisation Economic for Co-operation and Development (OECD) test guideline, TG 431 (ESAC 16-17 November 2006). Both test method development and multi-center study were performed using 0.63 cm(2) RHE tissue samples. The purpose of the present study was to demonstrate that similar results could be obtained using the validated test method adapted to 0.5cm(2) RHE tissue samples. Test method adaptation only consisted in applying a reduced volume of test substance (40 microL instead of 50 microL for liquids and 20 microL water+20mg test substance instead of 25 microL water+25mg test substance for solids) and a reduced propan-2-ol extraction volume (1.5 mL instead of 2 mL) during the MTT reduction assay. The test method was assessed with 25 representative test substances of different chemical classes. Among the latter, the 12 OECD reference test substances (6 corrosives and 6 non-corrosives) were evaluated and showed to be similarly classified as in vivo. More generally, the SkinEthic skin corrosion test adapted to 0.5 cm(2) RHE tissue samples fully complies with the OECD performance and reproducibility requirements with the 25 test substances.
 
Article
Dendritic cells (DC) are highly specialized antigen-presenting cells (APC) located in many non-lymphoid tissues and a specialized form of DC-the Langerhans cell (LC)-is found in the skin. The functionality of LC as APC is crucial for the induction of an allergic contact dermatitis. For a long time LC research has been hampered by the limiting numbers of functionally active LC that could be isolated from human skin. The addition of GM-CSF and IL-4 to the non-adherent fraction of mononuclear cells from peripheral blood generated a large amount of CD1a(+) HLA-DR(+) DC. These in vitro-generated DC exhibited the morphology, phenotype and autologous T-lymphocyte stimulating capacity of the human DC/LC system. We had tested phenotypical alterations of in vitro-generated DC under the influence of subtoxic concentrations of different chemicals and contact sensitizers. In vitro stimulation with the contact sensitizers urushiol, primin, C10-and C11-primin analogues, alantolactone, isoalantolactone and NiSO(4) resulted in a decrease of HLA-DR expression on the surface of these cells if the incubation period did not exceed 3 hr. Incubation with irritants like sodium lauryl sulfate (SLS) and benzalkonium chloride did not change or increase the HLA-DR surface expression under these conditions. With regard to the adhesion molecule ICAM-1, there was no clear difference between irritants and contact sensitizers. But based on the alteration of HLA-DR expression of dendritic cells under short-term exposure conditions, there was a clear-cut difference between irritants and contact sensitizers. In summary, this system can be used to discriminate between contact sensitizers and irritants.
 
Article
Colchicine is an alkaloid that has been widely used to treat gout. It also has a curative effect on cancer. Although many studies have shown that its effect on cell apoptosis was mediated by the activation of caspase-3, the pathways involved in the process remained obscure. Here we show some evidence regarding the missing information using human normal liver cells L-02 in our study. The effect of colchicine on apoptosis in L-02 cells and the apoptosis-associated signaling pathways were determined using different tests including cell viability assay, Annexin V and propidium idodide binding, PI staining, Hoechst 33342 staining, mitochondrial membrane potential assay, caspase activity assay and Western blot analysis. We found that colchicine-induced a dose-dependent drop of cell viability in L-02 cells; early apoptosis happened when cells were treated with 0.1μM of colchicine. The colchicine-induced loss of mitochondrial membrane potential, activation of caspase-3 and 9, up-regulation of Bax and down-regulation of Bcl-2 showed an evidence for the colchicine activity on apoptosis, at least, by acting via the intrinsic apoptotic pathway.
 
Article
Clivorine, a pyrrolizidine alkaloid, is a potent toxic compound extracted from a Chinese medicinal plant Ligularia hodgsonii Hook. We have previously shown that clivorine inhibited human normal liver L-02 cells proliferation and induced p38 mitogen-activated protein kinase phosphorylation. The aim of this study is to further observe the mechanism of clivorine-inhibited L-02 cells growth. In this paper we show here that clivorine can induce apoptosis in L-02 cells, reduce the expression of p53 protein but has no effect on the expression of Bcl-2 protein, and clivorine also induces the cleavage of the poly(ADP-ribose) polymerase in L-02 cells. Our results suggest that the anti-proliferative function of clivorine in L-02 cells may be due to its inducing cell apoptosis, and clivorine-induced cell apoptosis is independent of p53 protein.
 
Article
SMP-028 is a new compound for treatment of asthma. Oral administration of SMP-028 to rats was associated with toxicological events in endocrine organs. These events mainly consisted of pathological changes in the adrenal gland, testis, prostate, seminal vesicle, ovaries, and uterus. In this study, we set to clarify whether SMP-028 inhibits steroidogenesis in primary culture cells obtained from rat endocrine organs in vitro. Adrenal cells, testicular cells, and ovarian cells were treated with SMP-028 and the production of steroid hormones, i.e., progesterone, aldosterone, corticosterone, total testosterone, and estradiol from these cells was measured by radioimmunoassay. We found that the production of progesterone from these cells treated with SMP-028 at 1 μM decreased to 16%-67% that of the control. These findings indicate that SMP-028 inhibits steroidogenesis in rat endocrine organs in vitro. Considering that free maximum concentration in rats treated with SMP-028 are higher than the IC50 values for the inhibition of steroidogenesis in vitro, it is therefore believed that the toxicological events seen in rats following treatment with SMP-028 are due to inhibition of steroidogenesis in vivo.
 
Article
In toxicological research, immortalized human hepatocytes provide a useful alternative to primary hepatocytes because interindividual variability in the expression of drug-metabolizing enzymes and drug transporters can largely be eliminated. However, it is essential that the cell line retain the original phenotype. The purpose of this study was to characterize a novel spontaneously immortalized human hepatocyte cell line, HC-04, with respect to the transcript and functional protein expression profile for the major drug-metabolizing enzymes and transmembrane transporters. HC-04 cells retained hepatocyte-specific function including albumin production and ornithine transcarbamoylase and glucose-6-phosphatase activity. Most of the major CYP forms were expressed at basal levels and responsive to inducing agents. In particular, CYP3A4 was expressed abundantly, and HC-04 cells were able to metabolize the CYP3A4 probe, midazolam, at a rate similar to primary human hepatocytes. Furthermore, the major human sulfotransferase and UDP-glucuronosyltransferase forms, as well as members of the ABC and SLC transporter superfamilies, nuclear receptors, and hepatic transcription factors were also expressed. HC-04 cells readily responded to standard hepatotoxicants that are dependent on CYP-mediated bioactivation, while another, tumor-derived cell line remained refractory to the drug challenge. Collectively, HC-04 cells provide a reliable, stable, and reproducible model for biomechanistic studies in drug toxicology.
 
Article
To evaluate the anti-apoptotic effects of Joongpoongtang 05 (JP05), a mixture of plant extracts, on a Neuro-2a (N2a) cell model of oxygen and glucose deprivation (OGD)/reperfusion (OGDR), a neuroblastoma cell injury model was induced by OGDR. This model allowed us to investigate cerebral ischemic changes and the protective effects of JP05. JP05 treatment significantly enhanced cell viability and reduced the levels of lactate dehydrogenase, nitric oxide, reactive oxygen species and the oxidants/antioxidants balance in neuronal cells as compared to the untreated OGDR group. Here, JP05 reduced OGDR-induced expressions of heme oxygenase-1 and nitric oxide synthase, which may contribute to the neuroprotection. JP05 also partially reversed the effects of OGDR on NF-κB and activated Akt production. Our findings suggest that JP05 confers neuroprotective effects via anti-apoptotic property against OGDR-induced free radical injury in N2a cells.
 
Article
The in vitro effects of 1,1-dimethylhydrazine (UDMH) on prostaglandin E(2) (PGE(2)) synthesis, chemiluminescence, phagocytosis, microbicidal activity and chemotaxis in murine enriched-macrophage populations were evaluated. PGE(2) synthesis by resident peritoneal macrophages and chemiluminescence by activated macrophages were markedly suppressed in the presence of UDMH; phagocytosis and microbicidal activity were slightly to moderately suppressed, and chemotaxis was not affected. Two of these functions (PGE(2) synthesis and chemiluminescence) reflect macrophage immunoregulatory properties, and the UDMH-induced abrogation of these functions may be related to the previously reported immuno-enhancing effects of UDMH.
 
Article
The cardiac depressant effects of carbon tetrachloride (CCl(4)) and 1,1,1-trichloroethane (CH(3)CCl(3)) were evaluated in cultured heart cells from neonatal rats. Heart cells were grown on glass coverslips and formed a confluent monolayer that beat spontaneously, rhythmically and in synchrony. Contractility was assessed by video-motion analysis. Stock solutions of CCl(4) or CH(3)CCl(3) were prepared in dimethylsulphoxide (DMSO) and aliquoted (final DMSO concentration 0.2%) into medium (M199 supplemented with 5% serum) immediately prior to perfusion across myocytes in an environmentally controlled chamber. CCl(4) and CH(3)CCl(3) had a negative chronotropic effect on myocytes by prolonging the relaxation phase of beating. Duration of the contraction phase of beating, and peak velocity of cell wall movement were not affected by these halocarbons. Beating was stopped by 2.5 mm-CCl(4) or 5 mm-CH(3)CCl(3), and washout of these compounds resulted in a resumption of beating activity. Increasing (3.6 mm) or decreasing (0.6 mm) the calcium concentration of the medium (normal = 1.8 mm) significantly affected the duration of contraction and relaxation phases of beating, but did not alter the concentration-dependent action of CCl(4). A positive chronotropic effect of isoproterenol was evident from 10(-9) to 10(-6)m, but contractility was depressed by isoproterenol concentrations greater than 10(-8)m in the presence of 750 mum-CCl(4). This study demonstrates the usefulness of cultured heart cells for assessing the cardiac depressant and sensitizing actions of halogenated hydrocarbons.
 
Article
Organochalcogens are important intermediates and useful reagents in organic synthesis. Recent data from our laboratory demonstrated that bis and tris-selenide alkene derivatives are attractive synthetic targets because of their chemio-, regio- and stereo-selective reactions. Since the erythrocytic delta-aminolevulinate dehydratase (delta-ALA-D) activity could be an important indicator of toxicity, this report investigated bis and tris-selenide alkene derivatives effects on blood delta-ALA-D in vitro. To investigate the mechanisms by which these compounds inhibit human blood delta-ALA-D activity, a thiol reducing agent or zinc chloride were used. 1,2-Bis-selenide alkene derivatives 1a (R=4-MeOC(6)H(4)), 1b (R=4-ClC(6)H(4)) and 1c (R=2,4,6-Me(3)C(6)H(2)) did not inhibit human blood delta-ALA-D activity. 1,1,2-Tris-selenide alkene derivative 2a (R=C(6)H(5)) was the most potent delta-ALA-D inhibitor. Compounds 2b (R=4-MeOC(6)H(4)) and 2c (R=4-ClC(6)H(4)) displayed similar inhibitory potency towards delta-ALA-D activity. Dithiothreitol, a hydrophobic SH-reducing agent, was able to restore and to protect delta-ALA-D activity inhibited by tris-selenide alkene derivatives. Conversely, ZnCl(2) did not alter the enzyme inhibition induced by tris-selenide alkene derivatives. From these findings we suggest that 1,1,2-tris-selenide alkene derivatives inhibited delta-ALA-D activity by an interaction with essential sulfhydryl groups for the enzyme activity.
 
Article
In recent years workers in our laboratory have shown that several industrial chlorinated aliphatic hydrocarbon solvents interfere with the transport of bile acids by hepatocytes and this interference may account for the raised concentration of serum bile acids that has been observed after occupational exposure to solvents. There has been concern about the effects on workers of a selective solvent used in degreasing electrical equipment, 1,1,2-trichloro-1,2,2-trifluoroethane (FC 113). However, this compound has not been investigated for effects on bile acid transport. Therefore we undertook the present study to examine the direct in vitro effects of FC 113 on uptake and efflux of bile acids by isolated rat hepatocytes. FC 113, at non-cytotoxic doses after a 20-min equilibration time, showed significant (P < 0.05) inhibitory effects on the initial rate of uptake of taurocholate (TC), whereas accumulation of TC over an extended incubation time was not affected. Kinetic analysis revealed a non-competitive inhibition of TC uptake as evidenced by a decline in V(max) and an unaltered K(m). The initial rate of efflux of TC and the continuous efflux of this model substrate from preloaded cells incubated with different doses of solvent were not significantly different from those of controls. However, the highest dose of solvent inhibited the process of efflux at the early time points. The data suggest that FC 113 interferes with bile acid transport in a reversible manner similar to that of the chlorinated aliphatic hydrocarbons. It would be expected, therefore, that this solvent would cause an increase in serum bile acids in exposed workers.
 
Article
It has previously been shown that trichloroethylene (TRI) and 1,1,2-trichloro-1,2,2-tri-fluoroethane (FC 113) interfere with the transport of bile acids by isolated human and rat hepatocytes in a dose-dependent and reversible manner. This finding may explain the rise in serum bile acids (SBA) following exposure to these chemicals. However, the effect of these compounds on the transport of bile acids across the cellular membrane in the absence of confounding variables, such as interference by intracellular metabolism, binding to cytosolic proteins and intracellular conjugation, has not been investigated. Accordingly, in vitro effects of TRI and FC 113 on uptake of [(3)H]taurocholate (TC) into purified basolateral (blLPM) and canalicular (cLPM) rat liver plasma membrane vesicles were examined by a rapid filtration technique. Both TRI and FC 113 caused a dose-dependent inhibition of TC uptake into blLPM vesicles at an approximate concentration of 3 mm and 72 mum, respectively. Initial rates of TC uptake in the presence of TRI and FC 113 were significantly inhibited by 69 and 61%, respectively (P < 0.05). In contrast, these chemicals had no effect on TC uptake into cLPM vesicles. This confirms studies in intact cells where these solvents were found to inhibit the uptake of bile acids by hepatocytes rather than interfere with the process of efflux. In conclusion, and consistent with the previous findings, the data suggest that the mechanism of TRI and FC 113-induced elevation of SBA may, in part, be due to selective inhibition of bile acid transport by the parent compounds at the basolateral domain of the hepatocyte plasma membrane.
 
Article
1,10-Phenanthroline (phen) and metal-phen complexes display fungicidal and fungiststic activity, disrupt mitochondrial function and induce oxidative stress. We have examined the effect of these drugs on the structure of yeast and mammalian cell organelles and the integrity of cellular DNA. Exposure of Candida albicans to [Mn(phen)2(mal)].2H2O or [Ag2(phen)3(mal)].2H2O (mal H2 = malonic acid) resulted in DNA degradation whereas exposure to phen or [Cu(phen)2(mal)].2H2O did not. All drugs induced extensive changes to the internal structure of yeast cells including retraction of the cytoplasm, nuclear fragmentation and disruption of the mitochondrion. In the case of cultured mammalian cells [Cu(phen)2(mal)].2H2O induced apoptosis as evidenced by the ladder pattern of DNA fragments following gel electrophoresis and also the blebbing of the cell membrane. The other drugs produced non-specific DNA degradation in mammalian cells. In conclusion, phen and metal-phen complexes have the potential to induce apoptosis in fungal and mammalian cells. Given their distinct mode of action compared to conventional anti-fungal drugs, phen and metal-phen complexes may represent a novel group of anti-fungal agents for use either in combination with existing drugs or in cases where resistance to conventional drugs has emerged.
 
Article
Naphtho[1,2-b]furan-4,5-dione (NFD), a bioactive component of Avicennia marina, has been demonstrated to display anti-cancer activity. Activation of epidermal growth factor receptor (EGFR)-induced signaling pathway has been correlated with cancer metastasis in various tumors, including breast carcinoma. We use EGF as a metastatic inducer of MDA-MB-231 cells to investigate the effect of NFD on cell migration and invasion. NFD suppressed EGF-mediated protein levels of c-Jun and c-Fos, and reduced MMP-9 expression and activity, concomitantly with a marked inhibition on cell migration and invasion without obvious cellular cytotoxicity. NFD abrogated EGF-induced phosphorylation of EGF receptor (EGFR) and phosphatidylinositol 3-kinase (PI3K)/Akt. The specific PI3K inhibitor, wortmannin, blocked significantly EGF-induced cell migration and invasion. Furthermore, the EGFR inhibitor AG1478 inhibited EGF-induced MMP-9 expression, cell migration and invasion, as well as the activation of PI3K/Akt, suggesting that PI3K/Akt activation occur downstream of EGFR activation. These findings suggest that NFD inhibited the EGF-induced invasion and migration of MDA-MB-231 cells via EGFR-dependent PI3K/Akt signaling, leading to the down-regulation of MMP-9 expression. These results provide a novel mechanism to explain the role of NFD as a potent anti-metastatic agent in MDA-MB-231 cells.
 
Article
Carbon tetrachloride and 1,2-dichloroethane (1,2DCE) were added in vitro to freshly prepared slices of rat liver and the time- and concentration-dependence of their toxic effects on several metabolic parameters determined. With each agent, the most sensitive effect was an increase of malondialdehyde production by a microsomal preparation isolated from the treated slices. The next most sensitive parameter was the inhibition of amino acid incorporation into slice proteins, followed by inhibition of net K(+) accumulation and the induction of early necrotic changes, as indicated by loss of histological staining with azure II. Substantially greater exposures were required to reduce cellular ATP and to initiate entry of Ca(2+). This sequence was similar with both agents, but CCl(4) was the more potent in each case. When added in combinations of submaximally effective concentrations, the two agents produced at least additive inhibitions of protein synthesis and K(+) accumulation. We conclude that metabolic effects in liver slices can be a useful in vitro test for potential toxicity of chlorinated hydrocarbons. Amino acid incorporation and K(+) transport are the most convenient indicator systems, combining considerable sensitivity to relatively low levels of exposure with convenience of measurement.
 
Article
3-Monochloro-1,2-propanediol (MCPD) is a well-known by-product of acid-hydrolyzed soy sauce during its manufacturing process. MCPD has been reported genotoxic in vitro, and reproductive toxicity and carcinogenicity in rats. To evaluate the immunomodulatory effect of MCPD on murine splenocyte and macrophage in vitro, we investigated splenocyte blastogenesis by concanavalin A (Con A), anti-CD3, and lipopolyssacharide (LPS), the production of cytokines from splenocyte, and the activity of mouse peritoneal macrophages. There was a significant decrease in lymphocyte blastogenesis to Con A or anti-CD3 at subtoxic dose of MCPD. A significant decrease in splenocyte blastogenesis to LPS was also observed. The production level of interferon (IFN)-gamma on splenocyte culture with Con A was significantly reduced at the higher concentration than 1.0mM of MCPD. The levels of interleukin (IL)-4 and IL-10 were also decreased at high concentrations of MCPD. There was a significant decrease in production of nitric oxide (NO) by peritoneal macrophages treated with MCPD. MCPD also inhibits tumor necrosis factor (TNF)-alpha production of stimulated macrophages. These results indicate that MCPD might be able to reduce the functionality of lymphocytes and peritoneal macrophages in vitro.
 
Article
Naphtho[1,2-b]furan-4,5-dione (NFD), prepared from 2-hydroxy-1,4-naphthoquinone and chloroacetaldehyde in an efficient one-pot reaction, exhibits an anti-carcinogenic effect. NFD-induced apoptosis in MDA-MB-231 cells, as indicated by the accumulation of sub-G1 population, externalization of phosphatidylserine, loss of mitochondrial membrane potential (DeltaPsim) with subsequent release of cytochrome c, and activation of both capase-9 and caspase-3. This correlated with up-regulation in Bax and Bad, and down-regulation of various anti-apoptotic proteins, including Bcl-2, Bcl-X(L), Mcl-1, and survivin in NFD-treated cells. In the analysis of signal transduction pathway, NFD suppressed the phosphorylation of JAK2 in MDA-MB-231 cells without altering the expression of JAK2 protein. Activation of STAT3, Src, and PI3K/Akt were also inhibited by NFD. Moreover, the JAK2 inhibitor AG490 blocked JAK2, STAT3, Src, PI3K, and Akt activation, whereas both Src inhibitor PP2 and PI3K inhibitor wortmannin did not affect JAK2 activation. This suggests that STAT3, Src, and PI3K/Akt are downstream molecules of the JAK2 signaling pathway. AG490 treatment also mimics the cytotoxic effects of NFD. Taken together, these results indicate that NFD disrupts JAK2 pathway and induces apoptosis in MDA-MB-231 cells.
 
Article
Currently there is a great deal of interest in the study of natural compounds with free radical scavenging activity because of their potential role in maintaining human health and preventing diseases. In this paper, we report the antioxidant and cytoprotective properties of 4-(2-hydroxypropan-2-yl)-1-methylcyclohexane-1,2-diol (HPMCD) isolated from the aqueous extract of Decalepis hamiltonii roots. Our results show that HPMCD is a potent scavenger of superoxide (O(2)(*-)), hydroxyl ((*)OH), nitric oxide ((*)NO), and lipid peroxide (LOO(*)) physiologically relevant free radicals with IC(50) values in nmolar (56-582) range. HPMCD also exhibited concentration dependent secondary antioxidant activities like reducing power, metal chelating activity, and inhibition of protein carbonylation. Further, HPMCD at nmolar concentration prevented CuSO(4)-induced human LDL oxidation. Apart from the in vitro free radical scavenging activity HPMCD demonstrated cytoprotective activity in primary hepatocytes and ehrlich ascites tumor (EAT) cells against oxidative stress inducing xenobiotics. The mechanism of cytoprotective action involved maintaining the intracellular glutathione (GSH), scavenging of reactive oxygen species (ROS), and inhibition of lipid peroxidation (LPO). Based on the results it is suggested that HPMCD is a novel bioactive molecule with health implications in both prevention and amelioration of diseases involving oxidative stress as well as in the general well being.
 
Article
Naphtho[1,2-b]furan-4,5-dione (NFD), prepared from 2-hydroxy-1,4-naphthoquinone and chloroacetaldehyde in an efficient one-pot reaction, exhibits anti-carcinogenic effect. The results of present study showed that NFD inhibited the proliferation of breast cancer MDA-MB-231 cells through the induction of S-phase arrest and apoptosis. NFD-induced S-phase arrest was associated with a marked decrease in the protein expression of cyclin A, cyclin B, and cyclin-dependent kinase (Cdk)2. NFD-induced apoptosis was characterized by increase of sub-G1 population, phosphatidylserine (PS) externalization, and activation of caspases. Moreover, up-regulation of Bad and down-regulation of Bcl-2, Bcl-X(L), and survivin led to the loss of mitochondrial membrane potential (DeltaPsim), the release of cytochrome c and sequential activation of caspase-9 and caspase-3. NFD activated c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-regulated kinase (ERK) in MDA-MB-231 cells. Inhibitors of JNK (SP600125) and ERK (PD98059), but not p38 MAPK (SB203580) suppressed NFD-induced S-phase arrest and apoptosis in MDA-MB-231 cells. Both SP600125 and PD98059 attenuated Bad up-regulation, and reversed down-regulation of Bcl-2, Bcl-X(L), survivin, cyclin A, cyclin B, and Cdk2 in NFD-treated cells. Taken together, our data show that JNK and ERK-signaling pathways play important roles in NFD-mediated S-phase arrest and apoptosis of MDA-MB-231 cells.
 
Article
Six compounds, all newly synthesized triazole-pyrimidine derivatives that proved inhibitory of in in vitro growth of epimastigotes in Trypanosoma cruzi and of promastigotes of Leishmania donovani and Phytomonas staheli, were studied to investigate their toxic effects. As a biological model, the plant trypanosome P. staheli, which causes sudden wilt in the oil palm and Hartrot in the coconut palm, was used. The six compounds markedly inhibited macromolecule synthesis (nucleic acids and proteins) by the parasite. The cells treated with these compounds present severe damage in their ultrastructure-intense 'vacuolization, and appearance of lysosomes as well as other residual bodies. The mitochondrial section appeared larger in size. with a swollen matrix. In addition, these compounds changed the excretion of end metabolites, primarily affecting ethanol and acetate excretion, possibly by directly influencing certain enzymes (alcohol dehydrogenase and acetate synthetase) or their synthesis. 2000 Elsevier Science Ltd.
 
Article
Neurite outgrowth is an important aspect of neuronal plasticity and regeneration after neuronal injury. In this study we aimed to investigate the possible effect of 3-thiomethyl-5,6-dimethoxyphenyl-1,2,4-triazine (TDMT) on H(2)O(2)-induced impairment of neurite outgrowth. We found that TDMT could improve neurite outgrowth and neurite complexity in H(2)O(2)-exposed PC12 cells. Moreover, we found elevated levels of Hsp-70 and suppressed level of Hsp-90 in TDMT-treated cells in the presence of H(2)O(2). As another important signaling pathways that play role in neuritogenesis, as well as apoptosis, we measured the level of phosphorylated and total MAPKs proteins, JNK, ERK and p38 MAPK. We found that TDMT inhibits oxidative stress-induced phosphorylation of MAPKs. Since HSPs and MAPKs are both involved in coping with environmental changes, it will not be surprising if they can modify or augment each other's activity. Neuroprotective effect of this compound could represent a promising approach for treatment of neurodegenerative diseases.
 
Article
The generation, transmission (e.g. power lines, transformers, service wires, and electrical panels), and use (e.g. home appliances, such as electric blankets, shavers, and televisions) of electrical energy is associated with the production of weak electric and magnetic fields (EMF) which oscillate 50 (Europe) or 60 (USA) times per second (power-line frequency), falling in the extremely-low frequency (ELF) region of the electromagnetic spectrum. Epidemiological reports suggest a possible association between exposure to ELF-EMF and an increased risk of cancer (e.g. childhood acute leukaemia). Benzene is an established human leukomogen. This xenobiotic, which is unlikely to be the ultimate carcinogen, is metabolized in the liver to its primary metabolite phenol, which is hydroxylated to hydroquinone (1,4-benzenediol) and 1,2,4-benzenetriol. In this in vitro approach, to test the genotoxic and / or co-genotoxic potency of ELF-EMF, the cytokinesis block micronucleus (MN) method with Jurkat cells has been used. A 50 Hz magnetic field (MF) of 5 mT field strength was applied for different length of time (from 1 to 24 h), either alone or with benzene, 1,4-benzenediol, or 1,2,4-benzenetriol. Our preliminary results show that, after 24 h exposure, the frequency of micronucleated cells in MF-exposed cultures is 1.9 fold higher than in sham-exposed (control) cultures. Benzene exposure does not show any cytogenetic activity, whereas 1,4-benzenediol or 1,2,4-benzenetriol alone significantly affect the number of MN in Jurkat cells, as compared to untreated cultures. Moreover, co-exposure to ELF-MF does not seem to affect the frequency of micronuclei induced by benzene, 1,4-benzenediol, or 1,2,4-benzenetriol.
 
Article
The metabolism of the aromatic amine 1,3-diaminobenzene (MPD) was studied in vitro by use of the perfused rat liver, primary rat hepatocyte cultures and hepatic rat microsomes. The metabolites formed were compared with urinary metabolites excreted by the rat in vivo. Metabolites of [(14)C]MPD excreted by the perfused liver were found to be identical with urinary excreted metabolites in vivo, three of which were found to correlate with the N-acetylated derivatives N,N'-diacetyl-1,3-diaminobenzene, N,N'-diacetyl-2,4-diaminophenol and N-acetyl-1,3-diaminobenzene. Also, the primary hepatocyte cultures formed the metabolites found in the urine. However, several other metabolites, including some glucuronic acid conjugates, could be detected in the culture medium. In contrast to the perfused liver and the primary hepatocyte cultures, a reconstituted microsomal system was unable to form any of the metabolites formed by the other in vitro models and in vivo. The results show that in addition to the metabolites that can be detected in the urine in vivo, several other derivatives, including glucuronic acid conjugates of MPD, are formed by the rat liver. The results suggest that primary rat hepatocyte cultures are the preferred model system in metabolic studies in vitro and will constitute useful supplements to metabolic studies performed in the rat in vivo.
 
Article
The metabolism and toxicity of 1,3-dinitrobenzene(1,3-DNB) were examined in rat testicular cells that had been cultured for various amounts of time. The three cell systems utilized were: freshly isolated suspensions of Sertoli/germ cells; the same Sertoli/germ cells co-cultured for 24 hr; and Sertoli cell-enriched monolayers derived from the co-cultures and cultured for 96 hr. Indicators of toxicity were MTT reduction, neutral red incorporation, cellular ATP levels and lactate secretion into the media. 1,3-DNB (5-50 mum) caused a significant concentration-dependent decline in cellular ATP levels in the fresh cell suspension, but not in the cells that had been cultured for longer. No changes were observed either in MTT reduction or neutral red incorporation. Increased secretion of lactate into the media also did not prove to be a sensitive indicator of toxicity. Interestingly, 1,3-DNB metabolism to nitroaniline, nitroacetanilide and a covalently bound species was two to three times greater in the fresh cells, compared with either the 24- or 96-hr cell cultures. The data indicate that time in culture may have significant effects on both the capacity of testicular cells to metabolize 1,3-DNB and susceptibility to toxicity.
 
Article
The testicular toxicity of 1,3-dinitrobenzene (DNB) has been modelled in primary cultures of rat testis. The morphological response obtained in vitro following direct addition of the compound to the cultures was analogous to that seen in vivo. This was characterized by Sertoli cell vacuolation and germ cell detachment. The effect could be quantified using germ cell exfoliation into the culture medium, with a significant response occurring at toxicologically relevant concentrations of DNB (5 x 10(-6)m and above). Both Sertoli-germ cell co-cultures and Sertoli cell cultures were shown to be capable of xenobiotic metabolism, with nitroreduction of DNB being the predominant route. It is postulated that DNB or a Sertoli cell metabolite (probably an intermediate of nitroreduction produced in situ) is responsible for the testicular damage observed following administration of the compound in vivo.
 
Article
1,3-Dichloro-2-propanol (1,3-DCP) is a chlorinated compound used in the fabrication of industrial products such as hard resins, celluloid or paints. It has also been detected in instant soups and soy sauce. 1,3-DCP has been associated with major necrosis of the liver in humans [Chem.-Bio. Interact. 80 (1991) 73]. In humans and laboratory animals, 1,3-DCP is metabolised to dichloroacetone (1,3-DCA) by cytochromes P450 2E1 and 1A2 [J. University Occup. Environ. Health 14 (1992) 13]. 1,3-DCA is a hepatotoxin. We suggest that 1,3-DCA could be embryotoxic at doses that do not cause adverse maternal hepatic damage. To investigate the embryotoxic effects of 1,3-DCA, we have adapted a micromass culture method from Atterwill and colleagues [1992. A tiered system for in vitro neurotoxicity testing. In: Zbinden, G. (Ed.), The Brain in Bits and Pieces. Verlag M.T.C., Vollikon, pp. 89-91], using chick midbrain cells and from Wiger et al. [Pharmacol. Toxicol. 62 (1988) 32] using chick mesenchymal cells. The basis of the micromass system is that embryotoxins in vitro are likely to affect development and differentiation of disaggregated neuronal and limb bud micromass cultures. The endpoints chosen for the midbrain assay are resazurin reduction (viability), total protein content (cell number), morphological quantification of neuronal cultures (neuronal projection number) and of limb bud cultures (cartilage nodule number). Preliminary results using chick whole embryo cultures indicated that 1,3-DCA had an inhibitory effect on whole chick embryo development. We also found that embryonic derived cells were sensitive to 1,3-DCA but not 1,3-DCP at concentrations above 1 microM, suggesting a potential teratogenic effect of 1,3-DCA. The exposure to 1,3-DCP is not limited to industrial settings, and hence a better knowledge of its effects and tissue specific actions on embryonic-derived cells would be beneficial.
 
Article
Treatment of chick embryo hepatocytes in ovo and in culture with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in a dose-dependent induction of microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) activities. Significant induction was observed in the hepatocytes at TCDD doses as low as 10(-11) mol/egg (in ovo) and 10(-10)m (in culture). In contrast, 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) was a relatively weak inducer of these activities and only 10-20% of the induction responses observed for TCDD were elicited by MCDF at doses of 10(-6) mol/egg or 10(-7)m in culture. Co-treatment of the chick embryo hepatocytes with TCDD (10(-10) mol/egg in ovo; 10(-10)m in culture) and different concentrations of MCDF (10(-6) to 10(-8) mol/egg in ovo and 10(-7) and 10(-8)m in culture) resulted in minimal inhibition of TCDD-induced enzyme activities in ovo and a 37 to 50% inhibition in culture. The partial antagonist activity of MCDF in the chick embryo hepatocytes in culture paralleled the interactive effects previously reported in rodent liver and transformed rodent cell lines. TCDD (10(-7) to 10(-10)m) also caused an accumulation of hepta- and octacarboxyporphyrins in chick embryo hepatocytes (10(-7) to 10(-9)m); however, MCDF (10(-6) and 10(-5)m) elicited similar responses and MCDF did not significantly decrease the TCDD-induced porphyrogenic response in these cells. These results suggest that chick embryo hepatocytes in culture will serve as a useful model for investigating TCDD-induced gene transcription and the effects and mechanism of action of antagonists.
 
Article
It is increasingly recognised that mitochondria are potential targets to pharmacological and toxicological actions of membrane-active agents, including some 1,4-dihydropyridines derivatives (DHPs). The 5-acetyl(carbamoyl)-6-methylsulfanyl-1,4-dihydropyridine-5-carbonitriles (OSI-1146, OSI-3701, OSI-3761, and OSI-9642) is a new group of DHPs with minor differences on the molecular structure. It has also been shown that OSI-1146 displays cardiovascular, antioxidant, and antiradical activities, whereas OSI-3701 and OSI-3761 display hepatoprotective activity. Due to their protective properties, this group of DHPs may be potentially useful for the treatment of several pathological processes, including those associated with oxidative stress. However, the cellular targets for their pharmacological actions have not been investigated. The presented study, using isolated rat liver mitochondria was designed to investigate if mitochondria are a cellular target for the pharmacological and/or toxicological actions of these new group of DHPs. We studied the direct influence of these DHPs on rat liver mitochondrial function [bioenergetics, membrane permeability transition (MPT), and oxidative stress]. It was shown that OSI-1146, OSI-3761, and OSI-9642, in the concentration range of up to 200 microM, interfered with mitochondrial bioenergetics by affecting complexes I and II of the mitochondrial respiratory chain, the ATPase activity, and mitochondrial inner membrane permeability to protons. However, the effects of OSI-1146 were higher than those of OSI-3761 and OSI-9642. The remaining compound, OSI-3701, had no effect on the bioenergetic parameters tested. All the compounds increased the susceptibility of mitochondria to MPT, but, OSI-3701, not affecting the bioenergetic parameters, was the most potent. Moreover, all the compounds protected mitochondria against lipid peroxidation induced by the oxidant pair ADP/Fe(2+), but OSI-1146 was also the most potent. In conclusion, our results indicate that mitochondria are the potential intracellular targets for both protective and toxicological actions of the DHP compounds studied, suggesting that the potential use of these compounds as therapeutic agents should carefully consider their toxic effects to mitochondria.
 
Article
In animal models, naphthalene toxicity has been studied in different target organs and has been shown to be gender-dependent and metabolism related. In humans, it is readily absorbed and is metabolised by several cytochrome P450's. Naphthalene and its metabolites can cross the placental barrier and consequently may affect foetal tissues. The aim of this study was to compare the in vitro toxicity of naphthalene and its metabolites, 1-naphthol, 2-naphthol and 1,4-naphthoquinone, on human haematopoietic foetal progenitors (CFU-GM) derived from newborn male and female donors. The mRNA expression of Cyp1A2 and Cyp3A4 was also evaluated. Naphthalene did not affect CFU-GM proliferation, while 1-naphthol, 2-naphthol and particularly 1,4-naphthoquinone strongly inhibited the clonogenicity of progenitors, from both male and female donors. mRNA of Cyp1A2 and Cyp3A4 was not expressed neither at the basal level, nor after naphthalene treatment, while treatment with 1,4-naphthoquinone induced expression of both enzymes in both genders, with Cyp1A2 being expressed four times more than Cyp3A4. Female CFU-GM was significantly more sensitive to 1,4-naphthoquinone than male and after treatment both enzymes were expressed twice as much as in the male precursors. These results suggest that a gender-specific 1,4-naphthoquinone metabolic pathway may exist, which gives rise to unknown toxic metabolites.
 
Article
Quinones have diverse pharmacological properties including antibacterial, antifungal, antiviral, anti-inflammatory, antipyretic and anticancer activity. The cytotoxic potential of 1,4-naphthoquinone (NQ14) was studied against B16F1 melanoma cells grown in vitro. NQ14 treatment resulted in a concentration-dependent cytotoxicity as indicated by MTT assay and lactate dehydrogenase leakage assay. Depletion in cellular glutathione levels after 1h incubation with NQ14 correlated with the corresponding increase in reactive oxygen species generation as determined by 2',7'-dicholorofluorescein diacetate assay suggests the role of oxidative stress in cell death. The frequency of micronucleated binucleate cells increased with increasing doses of NQ14 with a corresponding decrease in the cytokinesis block proliferation index indicating the drug induced genotoxicity and cell division delay. Further, a dose-dependent decrease in the clonogenic cell survival indicated the potential of NQ14 to inhibit cell proliferation contributing to cell death. The cell death after NQ14 treatment may be attributed to apoptosis as seen in DNA ladder pattern along with necrosis as indicated in flow cytometric analysis of Annexin V/PI stained cells. Results of the present study demonstrate the cytotoxic and genotoxic potential of NQ14 by the induction of oxidative stress mediated mechanisms leading to tumor cell kill.
 
Article
1,4-Dihydropyridines (DHPs) used in the treatment of cardiovascular diseases, are calcium channel antagonists and also antioxidant agents. These drugs are metabolized through cytochrome P(450) oxidative system, majority localized in the hepatic endoplasmic reticulum. Several lipophilic drugs generate oxidative stress to be metabolized by this cellular system. Thus, DHP antioxidant properties may prevent the oxidative stress associated with hepatic biotransformation of drugs. In this work, we tested the antioxidant capacity of several synthetic nitro-phenyl-DHPs. These compounds (I-IV) inhibited the microsomal lipid peroxidation, UDPGT oxidative activation and microsomal thiols oxidation; all phenomena induced by Fe(3+)/ascorbate, a generator system of oxygen free radicals. As the same manner, these compounds inhibited the oxygen consumption induced by Cu(2+)/ascorbate in the absence of microsomes. Furthermore, compound III (2,6-dimethyl-4-(4-nitrophenyl)-1,4-dihydropyridin-3,5-ethyl-dicarboxylate) and compound V (N-ethyl-2,6-dimethyl-4-(4-nitrophenyl)-1,4-dihydropyridin-3,5-methyl-dicarboxylate) inhibited the microsomal lipid peroxidation induced by Nitrofurantoin and naphthalene in the presence of NADPH. Oxidative stress induced on endoplasmic reticulum may alter the biotransformation of drugs, so, modifying their plasmatic concentrations and therapeutic effects. When drugs which are activated by biotransformation are administered together with antioxidant drugs, such as DHPs, oxidative stress induced in situ may be prevented.
 
Article
Reactive metabolites of benzene 1,4-benzoquinone and 1,4-hydroquinone exert their toxic effects through covalent and/or oxidative damage to DNA and proteins. Since minipigs have been proposed as a suitable model species in toxicological and pharmacological research, the aim of this study was to explore mechanisms by which catechol, 1,4-hydroquinone and 1,4-benzoquinone destroy cytochrome P450 (P450) and induce oxidative stress in minipig liver microsomes. Our second goal was to assess the usefulness of minipig liver microsomes as a model system for the testing of the production of oxidative stress by clinically relevant quinone-containing compounds, e.g. anthracyclines. Of the three benzene metabolites tested, the highest P450 destruction was caused by 1,4-benzoquinone. This destructive effect did not correlate with the production of hydroxyl radicals as measured by ESR spin trapping which was the highest in samples containing 1,4-hydroquinone. Our results confirm previous findings that 1,4-benzoquinone exerts its effect mainly by direct attack on macromolecules while 1,4-hydroquinone rather stimulates the production of reactive oxygen species. Doxorubicin stimulated the production of hydroxyl radicals and the destruction of P450 similarly as 1,4-hydroquinone. Minipig liver microsomes should be further tested as a possibly suitable model system for the testing of potential modulators of the toxicity of doxorubicin.
 
Article
9-Bromo-5-morpholino-tetrazolo[1,5-c]quinazoline (BMTQ) acted cytotoxically on murine leukemia cell line L1210 and human colon carcinoma cells Caco-2. We found the two highest concentrations of BMTQ (149.2 and 74.6 microM) induced an acute cytotoxic effect, however other tested concentrations (<74.6 microM) manifested a concentration/dependent and time/dependent cytotoxic effect. The sensitivity of murine leukemia cells L1210 and human colon carcinoma cells Caco-2 was expressed in the same order. The cytotoxicity of BMTQ was not accompanied by changes of the cell cycle profile. Following the cytotoxicity-related effects of BMTQ we observed the induction of ssDNA breaks after BMTQ treatment. All the concentrations of BMTQ increased the level of ssDNA breaks 1.3-2.9 times (after 2 h of treatment) and 1.6-2.8 times (after 4 h of treatment) in Caco-2 cells compared to the control. No apoptotic DNA fragmentation induced by BMTQ in Caco-2 cells was recorded.
 
Article
In Salmonella typhimurium mutagenicity assays the metabolic activation of 2-acetylaminofluorene (2AAF) was greater by isolated hepatocytes from two species resistant to 2AAF-induced hepatocarcinogenesis, the guinea-pig and rabbit, than by hepatocytes from the highly susceptible rat liver. S. typhimurium strain TA98/1,8DNP(6) was less sensitive to 2AAF-induced mutagenesis than strain TA98. Even in the absence of bacterial esterification (TA98/1,8DNP(6)), 2AAF mutagenicity was always lowest following activation by rat hepatocytes. Paraoxon (an inhibitor of deacetylase) inhibited by more than 90% the mutagenicity of 2AAF with rat and guinea-pig hepatocytes, but only partially inhibited the mutagenicity of N-hydroxy-2-acetylaminofluorene-a metabolite of 2AAF-particularly at low substrate concentrations. In a modified mutagenicity assay in which hepatocytes and 2AAF were pre-incubated before the addition of TA98, a reduction of 2AAF mutagenicity was observed with guinea-pig but not rat hepatocytes. Thus, even when the integrity of the cells used for metabolic activation is maintained, and when bacterial esterification is avoided, conventional Salmonella assays do not show a correlation between the mutagenicity of 2AAF in vitro and its carcinogenicity in vivo. The correlation may, however, be improved by modification of in vitro conditions.
 
Article
Homology modelling of the human microsomal epoxide hydrolase (EH) enzyme based on the fungal (Aspergillus niger) EH crystallographic template is reported. The active site lies in a well-defined, essentially hydrophobic, pocket within the overall enzyme structure. Two tyrosine residues, that are conserved in all known mammalian EH sequences, are able to form hydrogen bonds (one per tyrosine residue) with the epoxide oxygen atom on the known EH substrate, styrene oxide. There is also a small hydrophobic cleft, within the active site region, where the phenyl group of styrene oxide can bind, but this appears to be restricted such that the presence of bulky side-chains will render poor substrate status to the incoming epoxide molecule. Quantitative structure-activity relationship (QSAR) studies on a series of low molecular weight epoxides provide useful results which appear to be generally consistent with the human microsomal EH model, and thus may be used predictively for assessing the EH substrate and/or inhibitor status of untested compounds.
 
Article
The determination of a possible corrosive or irritative potential of certain products and ingredients is necessary for their classification and labeling requirements. Reconstructed skin as a model system provides fundamental advantages to single cell culture testing and leads to promising results as shown by different validation studies (for review: Fentem, J.H., Botham, P.A., 2002. ECVAM's activities in validating alternative tests for skin corrosion and irritation. ATLA 30(Suppl. 2), 61-67). In this study we introduce our new reconstructed epidermis "Epidermal-Skin-Test" (EST-1,000). This fully grown epidermis consists of proliferating as well as differentiating keratinocytes. EST-1,000 shows a high comparability to normal human skin as shown by histological and immunohistochemical data. Characteristic markers (KI-67, CK 1/10/5/14, transglutaminase, collagen IV, involucrin, beta 1 integrin) can be identified easily. The main focus of this work was to characterize EST-1,000 especially with respect to its barrier function by testing several substances of known corrosive potential. Skin corrosion was detected by the cytotoxic effect of the substances on a reconstructed epidermis after short-term application to the stratum corneum. The effect was determined by standard MTT assay and accompanying histological analysis. Hence EST-1,000 shows a very high predictive potential and closes the gap between animal testing and the established full-thickness model Advanced-Skin-Test 2,000 (AST-2,000) (Noll, M., Merkle, M.-L., Kandsberger, M., Matthes, T., Fuchs, H., Graeve, T., 1999. Reconstructed human skin (AST-2,000) as a tool for pharmaco-toxicology. ATLA 27, 302).
 
Article
Even though there have been some investigations into cellular responses induced by ultrafine titanium dioxide (TiO(2)) in vitro, the relationship between cellular responses and secondary particle size is still not clear. In this study, a stable and uniform TiO(2)-cell culture-medium dispersion was prepared, and cellular responses prompted by "ultrafine secondary particles" were examined. The TiO(2)-DMEM-FBS dispersion included secondary particles in which the secondary particle size was 100 nm or less. In the present study, a "secondary particle" was defined as a complex aggregate of TiO(2) primary particles, proteins from FBS and other medium components. Secondary particle size did not influence the cell viability. The TiO(2)-DMEM-FBS dispersion introduced to the human keratinocyte HaCaT cells caused weak intracellular oxidative stress and apoptosis. The cellular influence of ultrafine TiO(2)in vitro is caused by the following mechanisms: (1) Secondary particles are formed. Ultrafine TiO(2) particles dispersed in medium immediately form secondary particles with proteins and salts. (2) "Ultrafine" secondary particles are taken up by the cells. The secondary particles reach the cells by diffusion and/or sedimentation and are taken up by the cells, through endocytosis. (3) Intracellular reactive oxygen species (ROS) level increases. Internalized secondary particles induce an increase in intracellular reactive oxygen species levels, although the secondary particles do not break up in the cell. In the case of ultrafine TiO(2), the increase of the intracellular ROS level was minimal. Moreover, the antioxidation system of cells such as glutathione was working. (4) Apoptotic cell death is induced. An accumulation of oxidative stress activates the apoptotic pathway (such as the caspase-3) and subsequently induces apoptotic cell death. After 24h of exposure to TiO(2), the percentage of apoptotic cells was only 6-7%. As a result, although the ultrafine TiO(2) particles induce some cellular responses, these cellular responses to ultrafine TiO(2) are weaker than those of other cytotoxic ultrafine metal oxide particles, such as nickel oxide.
 
Article
Polychlorinated biphenyls (PCBs) are persistent pollutants in aquatic environments, often causing the decline or disappearance of wild populations. The primary aim of this study was to investigate the genotoxic effects of some PCBs (PCB153 (2,2',4,4',5,5'-hexachlorobiphenyl) and 138 (2,2',3,4,4',5'-hexachloro-biphenyl), both non-dioxin-like compounds, and the pentachlorobiphenyls PCB118 (2,3',4,4',5-) and 101 (2,2',4',5,5'-), the former an ortho-substituted, low-affinity dioxin-like compound and the latter a non-coplanar congener classified as non-dioxin-like) in fish cells (RTG-2). These congeners are mostly present in surface waters and in edible aquatic organisms and the loss of DNA integrity in vitro serves as a sensitive biomarker of cytogenetic alterations and is considered as an initial step for the identification of genotoxic effects. The alkaline comet assay and the micronucleus test show clear genotoxic damage after short and longer exposure (2 and 24h) to maximum soluble, non-cytotoxic doses, evident sooner with PCBs 101 and 118. Oxidative stress situations involving ROS release, reduction in total GSH, lipid peroxidation and alteration to superoxide dismutase, seen after exposure with all the congeners, though with different kinetics, seem the most likely explanation for the genotoxic damage. This appears to be confirmed by the modified comet assay (pH 10) for detection of oxidized bases using endonuclease III. The increased generation of intracellular ROS might explain the apoptosis seen after treatment with the single PCBs and evaluated on the basis of the rise in 3-7 caspase activity. Therefore both the non-coplanar, non-dioxin-like PCBs (153, 138, 101) and the low-affinity dioxin-like compound PCB118 cause evident genotoxic damage, probably as a consequence of oxidative stress.
 
Article
In this study, HepG2 cells were exposed to 0.04-40mg/L Irgarol 1051. Results show that Irgarol 1051 can damage cell morphology and cause a significant decrease in cell viability. Positive staining by Annexin V, caspase-3 activity enhancement, and the damage in cell ultrastructure indicated an apoptotic mode of cell death for 4.0mg/L Irgarol 1051 treatment. At the same time, capase-9 was also significantly induced by 0.4 and 4.0mg/L Irgarol 1051 at 72h, which suggests that the intrinsic mitochondria pathway was involved in the apoptosis. The mitochondrial membrane potential decreased significantly after the HepG2 cells were exposed to Irgarol 1051 for 6 and 72h. Especially, the translocation of cytochrome c from mitochondria to cytosol was recorded, supporting the idea that the mitochondrial pathway was involved in the apoptosis signal pathways induced by Irgarol 1051. The significantly increased levels of intracellular reactive oxygen species (ROS) and an immediate ROS burst were also recorded. The results here may imply that Irgarol 1051 induces HepG2 cell apoptosis through mitochondrial dysfunction and oxidative stresses. Although it is possible that this chemical has no detrimental effects on human health at the environmentally relevant concentration, it may cause problems to top coastal predators due to bio-accumulation through the food chain.
 
Article
The aminothiols cysteamine (CSM) and 2-[(aminopropyl)amino] ethanethiol (WR-1065) are radioprotectors, in that they reduce the mutagenic and potentially lethal consequences of ionizing radiation. We have studied the effects of these compounds on the induction of gene conversion at the trp5 locus in Saccharomyces cerevisiae strain D7 by two chemical mutagens: bleomycin (BLM) and beta-propiolactone (beta-PL). Both mutagens are potent recombinagens in this assay, giving rise to trp5 convertant frequencies greater than 10(-3). CSM and WR-1065 are effective antimutagens, reducing the recombinagenic effect of beta-PL by about 95%. The maximum reduction in the recombinagenic activity of BLM was 91% with CSM and 89% with WR-1065. Although the antimutagenic effects of the aminothiols against beta-PL and BLM appear to be similar, they stem from different underlying mechanisms. Aminothiols protect against the recombinagenicity of beta-PL through direct inactivation of the electrophilic mutagen by the nucleophilic antimutagen. They protect against that of BLM through modification of physiological conditions, most notably by depletion of oxygen required for BLM activity.
 
Article
Pseudomonas aeruginosa is an important opportunistic pathogen of the human urinary bladder. Similar to rat liver S9, the cell-free extract from P. aeruginosa caused significant increase of histidine reversion numbers with the Salmonella typhimurium tester strain TA98 in the Ames Salmonella mutagenicity assay in the presence of either 2-aminofluorene, 4-aminobiphenyl, or benzidine procarcinogens. The presence of cytochrome P-450 protein in the cell-free extract was demonstrated by the carbon monoxide difference spectrum. We employed gene knockout technology to inactivate one of the three known putative cytochrome P-450 genes of P. aeruginosa, namely CYP107S1, which we postulated to be the most likely to induce activation. The ampicillin resistant gene from PUC19 DNA confers carbenicillin resistance to P. aeruginosa. We inserted a synthetic ampicillin gene flanked by 40 base-pairs of the 5' and 3' untranslated region of the CYP gene by electroporating the synthetic gene into electrocompetent P. aeruginosa cells. CYP107S1 knockout strains were selected on 1000 microg/ml carbenicillin plates. A single cloned carbenicillin resistant colony was isolated and used to determine its mutagenic capacity using Ames Salmonella mutagenicity assay. The results showed that Salmonella TA98 tester strain returned the number of revertants to its baselines level indicating the lack of metabolic activation of procarcinogens in the P. aeruginosa CYP107S1 knockout cell-free extract. In addition, the characteristic cytochrome P-450 peak determined by the carbon monoxide difference spectrum was completely absent in the cell-free extract from this CYP107S1 knockout strain bacterium. Homologous recombination of the synthetic ampicillin gene on the CYP 107S1 P-450 locus was confirmed by PCR on purified genomic DNA extracted from the knockout bacterium. The metabolic activation of tested procarcinogens is, therefore, carried out by CYP107S1 in P. aeruginosa.
 
Article
Short Time Exposure (STE) test is an easy in vitro eye irritation test that assesses cytotoxicity in SIRC cells (rabbit corneal cell line) following a 5 min dose treatment. To assess intra-laboratory reproducibility, medium control, three vehicles (saline, saline containing 5% (w/w) dimethyl sulfoxide, and mineral oil) and three standard chemicals (sodium lauryl sulfate, calcium thioglycolate, and Tween 80) were evaluated. Assessments were repeated 30 times for vehicles and 18 times for standard chemicals; resulting in almost the same cell viability and a low coefficient of variation value. In addition, the STE eye irritation rankings of three standard chemicals, as calculated on the cell viabilities in 5% and 0.05% solutions were in agreement in all tests. Based on these results, high intra-laboratory reproducibility was confirmed. In addition, the irritation category (irritant and non-irritant) was evaluated for 109 chemicals with STE test, globally harmonized system (GHS) classification, and European Union (EU) classification. The results of the evaluation found the STE classification to have an accuracy with GHS classification of 87% and with EU classification of 83%, which confirmed the excellent correspondence. The correspondence of STE rankings (1, 2, and 3) based on the prediction model by STE test with the eye irritation rankings by GHS (non-irritant, categories 2 and 1) and EU (non-irritant, R36, and R41) was 76% and 71%, respectively. Based on the above results, STE test was considered to be a promising alternative method for assessing eye irritation that has high intra-laboratory reproducibility as well as an excellent predictability of eye irritation.
 
Article
In vitro toxicity screening can reduce the attrition rate of drug candidates in the pharmaceutical industry in the early development process. The focus in this study is to compare the sensitivity for cytotoxicity of a time-resolved fluoro metric oxygen probe with that of a fluoro metric Alamar Blue™ (AB) assay. Both assays measure mitochondrial activity by either oxygen consumption (LUX-A65 N-1 (MitoXpress, Luxcel) probe) or NADH/FADH conversion (AB). Both assays were carried out with increasing concentrations of 109 reference compounds using rat H4IIE and human HepG2 hepatocytes at incubation periods of 24, 48 and 72 h. Prior to this study, the influence on medium with either glucose or galactose was studied to analyze the rate of glycolysis and oxygen consumption, which latter process may be impaired in hepatoma cells. Inhibitors of oxygen consumption in combination with a glucose up-take inhibitor showed the largest consumption rate differences in the presence of 5 mM of glucose.
 
Article
Moulds of the genus Alternaria are common contaminants of some food crops. Some isolates have been shown to produce mutagenic compounds called altertoxins. Altertoxin I (ATX-I) and altertoxin III (ATX-III) were examined for activity in the Raji cell Epstein-Barr virus early antigen (EBV-EA) induction system and in the C3H/10T1 2 murine fibroblast cell transformation system. Exposure of Raji cells to ATX-I or ATX-III activated EBV-EA expression by 8- and 9.5-fold, respectively. A single exposure of C3H/10T1 2 cultures to ATX-I or ATX-III resulted in significant increases in cell transformation, and the response to ATX-I was stronger. Both altertoxins enhanced the transformation of C3H/10T1 2 cells, and chronic exposure of non-initiated C3H/10T1 2 cells to ATX-I and ATX-III, starting 6 days after cells were plated, resulted in cell transformation in 8 59 and 12 37 dishes, respectively, compared with transformation in only 2 63 control dishes. Since activation of EBV-EA in Raji cells has been positively correlated with tumour promoters, these data together indicate that ATX-I and ATX-III are not just mutagens but have a potential role in cell transformation.
 
Article
The widely used plasticizer and rodent carcinogen di-(2-ethylhexyl) phthalate (DEHP) was examined for activity in the C3H 10T 1 2 murine fibroblast cell transformation system. Treatment with DEHP or its metabolite, mono-(2-ethylhexyl) phthalate, did not produce oncogenic transformation, initiate the process of transformation in cultures treated with a tumour promoter or promote the process of transformation in cultures pretreated with a chemical carcinogen. These findings are consistent with the suggestion that the carcinogenicity of DEHP is mediated by an indirect mechanism and not by covalent interaction of DEHP with DNA.
 
Article
We investigated whether the two-stage transformation assay can be applied in routine testing for promoter-like activity of cigarette smoke condensate (CSC) as an in vitro equivalent of an in vivo tumorigenicity assay (mouse skin painting). We adopted a published assay procedure (Frazelle et al., 1983a), using 3-methylcholanthrene (MCA, 0.37mumol/litre, 24hr treatment) as the initiator. Rigorously standardized experimental conditions, such as multiparameter-screened serum, one fixed subculture level, and a rigid preculturing schedule, were employed. Transformation was expressed as the fraction of dishes containing type II and type III foci. Compared to the positive control, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), transformation responses to CSC (three CSC batches that had different in vivo activity) were lower. Variations in dose-response relationships did not allow distinction between two of the three CSC batches, even with data pooled from seven assay repetitions over 2 years. In a second approach, to enhance the assay resolution, that is, the signal-to-noise ratio, promoter treatment twice per week was ineffective: the response and the background were both increased. Lowering the initiator concentration (0.08mumol/litre) enhanced the signal-to-noise ratio for TPA, but not for CSC. Even after standardization and enhancement of sensitivity, the two-stage transformation assay is useful primarily for qualitative assessment of promoter-like activity of weak promoters, such as CSC, rather than for quantitative comparisons.
 
Article
Various kinds of stress such as heat, UV, gamma-rays and chemicals that cause DNA damage induce heat shock proteins (Hsps), and in particular Hsp70. The Hsps cytoprotective function is not fully understood, although these proteins act as molecular chaperones or modulators of intracellular levels of reactive oxygen species (ROS). Recently, Hsps have been proposed to play a significant role in DNA repair after UV or gamma-ray irradiation. Ionizing radiation targets DNA molecules either via direct interaction or via production of free radicals and ROS. When exposed to gamma-rays C3H 10T1/2 cells are radiosensitive, therefore we decided to use them to investigate Hsp induction after ionizing radiation and their protective role against DNA damage. Here we demonstrate the induction of Hsps by gamma-rays, and investigate the kinetics of expression after irradiation at different doses. We also show that Hsp70 overexpression acts as a radioprotective mechanism towards the first event of DNA damage and increases long term viability. A preliminary investigation on the cell cycle does not evidence a significant protective action of inducible Hsp70 on it.
 
Article
CPT-11, a derivative of camptothecin (CPT) that interacts with type-I DNA topoisomerase, induced apoptosis in HL60 and Daudi cells in vitro. This cytotoxic activity was time and dose dependent, and was prevented by cycloheximide (CHX), a protein synthesis inhibitor, indicating the requirement of new protein synthesis for CPT-11-induced apoptotic cell death. Ac-Tyr-Val-Ala-Asp-aldehyde (YVAD) and Ac-Asp-Glu-Val-Asp-aldehyde (DEVD), synthesized tetrapeptide inhibitors of interleukin(1beta)-converting enzyme (ICE)- and CPP32/Yama-like proteases, were used to examine the CPT-11-induced death signal transduction. These inhibitors blocked CPT-11-induced cytotoxicity in a time- and dose-dependent manner. Cytotoxic activity of SN-38, an active metabolite of CPT-11, was about 1000-fold that of CPT-11 and was also prevented by CHX, YVAD and DEVD. The doses of YVAD, however, were a little too high; the prevention by YVAD is then thought to be non-specific. In addition, lymphocytes obtained from normal and lpr(cg) mutant mice showed similar susceptibility to CPT-11 cytotoxicity. These results indicate the direct involvement of CPP32/Yama-like protease in the CPT-11-induced death signal transduction pathway, and no involvement of Fas antigen in the pathway.
 
Top-cited authors
John J Schlager
  • Air Force Research Laboratory
Saber M Hussain
  • Wright-Patterson Air Force Base
Jeffery Gearhart
  • The Henry M. Jackson Foundation for Military Medicine
Horst Spielmann
  • Freie Universität Berlin
Harvey Babich
  • Yeshiva University