V79 Chinese Hamster Fibroblasts Research Articles

The effects of sodium diethyldithiocarbamate in fibroblasts V79 cells in relation to cytotoxicity, antioxidative enzymes, glutathione, and apoptosis

Sodium diethyldithiocarbamate (DETC) is the main metabolite of disulfiram. Recently, we reported that mechanism of disulfiram cytotoxicity in V79 cells might be partially connected with thiol redox-state imbalance. Here, we examined the effect of DETC on the level of intracellular glutathione (GSH), protein oxidation (measured as PC—protein carbonyl content), lipid peroxidation (measured as TBARS—thiobarbituric acid reactive substances), antioxidant enzymatic defense, as well as on apoptosis. We used V79 Chinese hamster fibroblasts cells with and without modulated glutathione (GSH) level by N-acetyl-l-cysteine (NAC). We showed that treatment with DETC at concentrations that cause a moderate increase in thiol-state imbalance but not cell death stimulates oxidative stress measured as increased level of PC and TBARS, adaptive response of GSH-related enzymes and apoptosis. Our results show that cellular effects of DETC are partially attributable to the initial redox cellular state, since the increase of GSH level by NAC pre-treatment prevented the observed changes.

Fruits and vegetables protect against the genotoxicity of heterocyclic aromatic amines activated by human xenobiotic-metabolizing enzymes expressed in immortal mammalian cells

Heterocyclic aromatic amines (HAAs) can be formed during the cooking of meat and fish at elevated temperatures and are associated with an increased risk for cancer. On the other hand, epidemiological findings suggest that foods rich in fruits and vegetables can protect against cancer. In the present study three teas, two wines, and the juices of 15 fruits and 11 vegetables were investigated for their protective effect against the genotoxic effects of 2-amino-3-methylimidazo[4,5- f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP). To closely mimic the enzymatic activation of these HAAs in humans, genetically engineered V79 Chinese hamster fibroblasts were employed that express human cytochrome P450-dependent monooxygenase (hCYP) 1A2 (responsible for the first step of enzymatic activation) and human N( O)-acetyltransferase (hNAT) 2*4 or human sulfotransferase (hSULT)1A1*1 (responsible for the second step of enzymatic activation): V79-hCYP1A2-hNAT2*4 for IQ activation and V79-hCYP1A2-hSULT1A1*1 for PhIP activation. HAA genotoxicity was determined by use of the comet assay. Black, green and rooibos tea moderately reduced the genotoxicity of IQ (IC 50 = 0.8–0.9%), whereas red and white wine were less active. From the fruit juices, sweet cherry juice exhibited the highest inhibitory effect on IQ genotoxicity (IC 50 = 0.17%), followed by juices from kiwi fruit, plum and blueberry (IC 50 = 0.48–0.71%). The juices from watermelon, blackberry, strawberry, black currant, and Red delicious apple showed moderate suppression, whereas sour cherry, grapefruit, red currant, and pineapple juices were only weakly active. Granny Smith apple juice and orange juice proved inactive. Of the vegetable juices, strong inhibition of IQ genotoxicity was only seen with spinach and onion juices (IC 50 = 0.42–0.54%). Broccoli, cauliflower, beetroot, sweet pepper, tomato, chard, and red-cabbage juices suppressed IQ genotoxicity only moderately, whereas cucumber juice was ineffective. In most cases, fruits and vegetables inhibited PhIP genotoxicity less strongly than IQ genotoxicity. As one possible mechanism of antigenotoxicity, the inhibition of activating enzymes was studied either indirectly with diagnostic substrates or directly by measuring CYP1A2 inhibition. Only sour cherry, blueberry, and black currant juices suppressed the first step of HAA enzymatic activation, whereas most plant-derived beverages inhibited the second step.

Sequential fractionation with concurrent chemical and toxicological characterization of the combustion products of chlorogenic acid

Chlorogenic acid is the most abundant polyphenol found in the tobacco plant. The biological effects of its combustion products remain largely unknown. In this study, chlorogenic acid was burned at 640 °C for 2 min and the particulate matter of the smoke was collected onto Cambridge filter pads followed by selective extraction in five different solvents. Various fractions of the chlorogenic acid combustion products were tested for induction of micronuclei in V79 Chinese hamster fibroblast cells. Over 40 compounds were identified in the dimethyl sulfoxide (DMSO) extract by high-performance liquid chromatography coupled to electrospray time-of-flight mass spectrometry (HPLC/TOF-MS). The DMSO extract was then fractionated into three major fractions by preparative LC. The fraction inducing the highest degree of toxicity was further separated into four sub-fractions. The sub-fraction responsible for the most toxic response was determined to contain catechol as its major component. The overall reproducibility of the combustion, the extraction procedure and the chemical characterization of the compounds responsible for the toxicity in the chlorogenic acid smoke were evaluated by LC/TOF-MS.

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

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

The 'Cosmic silence' experiment: on the potential adaptive role of environmental background radiation

In low dose radiobiology, there is a strong interest in phenomena that could imply a deviation from the linear-no-threshold (LNT) model, which is currently used to estimate the excess relative cancer risk at low doses of ionizing radiation (IR) via a linear extrapolation from high- and intermediate-dose epidemiology risk data. Such phenomena include the adaptive response as well as bystander effects , occurring in cells which do not experience direct radiation exposure. These phenomena suggest that biological response to harmful agents such as ionising radiation may be more complex than that assumed by the LNT model. In particular, environmental background radiation represents a source of chronic low dose-rate exposure which may condition the response of living systems to acute exposure to genotoxic agents, also including IR itself, via an adaptive response. To clarify this aspect is essential not only to better evaluate the risk of chronic occupational exposure, but also to understand the role of environmental background radiation on biological evolution. Reduction of background environmental IR dose rate exposure is achieved at the National Laboratories at Gran Sasso (LNGS) of the Italian Institute for Nuclear Physics (INFN). Located underneath the Gran Sasso mountain range in central Italy, these laboratories offer an excellent opportunity to continuously maintain cell cultures at extremely reduced levels of environmental background radiation. The Cosmic Silence experiment represents the natural extension, to a human cell culture model (TK6 lymphoblastoid cells), of earlier experimental studies on S. Cerevisiae (L. Satta et. al. Mut Res 1995) and V79 Chinese hamster fibroblasts (L. Satta et. al. Radiat Environ Biophys 2002), which indicated a progressive disappearance of an adaptive response in cells kept under reduced environmental background IR conditions. For the Cosmic Silence experiment, TK6 parallel cultures were set up in the underground LNGS laboratories and in Rome and planned to be maintained and assayed for 12 months. Starting at 6 months of continuous, parallel cultures, measurements of DNA damage (micronuclei) or oxidative metabolism (antioxidant enzymatic activities of SOD, GPx, and Catalase) indicate that significant deprivation of environmental background IR renders cells less tolerant to an acute X-ray exposure of 1-2 Gy. These results are compatible with the concept that environmental background IR is behaving as an adaptive agent.

Changes in antioxidant defense systems induced by thiram in V79 Chinese hamster fibroblasts

The role of antioxidant defence systems in protection against oxidative damage of lipids and proteins induced by fungicide thiram during in vitro exposure was investigated in cultured Chinese hamster V79 cells with normal, depleted, and elevated glutathione (GSH) levels. We analyzed the catalytic activities of superoxide dismutases (SOD1 and SOD2), Se-dependent and Se-independent glutathione peroxidases (GSH-Px), glutathione reductase (GR), and catalase (CAT), as well as total glutathione/glutathione disulfide ratio (GSH total/GSSG). Thiram treatment resulted in an increase in activities of SOD1, Se-dependent GSH-Px, and GR at the highest tested dose (150 μM). On the contrary, inhibition of CAT and Se-independent GSH-Px activities, and no significant changes in the level of SOD2 activity was observed at any tested doses (100–150 μM). GSH total/GSSG ratio in the 100 μM thiram treated cells was not significantly changed comparing to the control, despite significant decrease of GSH total (50%). In 150 μM thiram treated cells the ratio falls to 43% of control value. Pretreatment with l-buthionine sulfoximine ( l-BSO), an inhibitor of GSH synthesis, significantly enhanced decrease in CAT and Se-independent GSH-Px activities, as well as GSH total/GSSG ratio, and reduced Se-dependent GSH-Px activity, following exposure to thiram. Simultaneously, l-BSO pretreatment enhanced increase in SOD1 activity, and had no effect on SOD2, following thiram exposure. Pretreatment with N-acetyl cysteine (NAC), a GSH precursor, prevented enzymatic changes in CAT, Se-dependent GSH-Px, GR, SOD1 activities, and significantly decreased SOD2 activity following exposure to thiram. GSH total/GSSG ratio was restored to the control value. This study suggests that following the changes in antioxidant defense systems thiram can act through the production of free radicals.

Induction of micronuclei by ochratoxin A is a sensitive parameter of its genotoxicity in cultured cells

In order to better characterize the ochratoxin A (OTA)-induced DNA damage and to further investigate factors which may modulate dose-effect relationships in cells, the induction of micronuclei was studied in V79 Chinese hamster fibroblast cells and in primary cultures of porcine urothelial bladder epithelial cells (PUBEC). OTA was able to induce micronuclei in PUBEC and V79 cells at concentrations below those which were overtly cytotoxic. OTA concentrations between 0.03 and 1 μM caused a dose-dependent increase of micronuclei in V79 cells (up to 3-fold compared to controls); but the lowest tested concentration of 0.01 μM OTA did not induce a higher frequency of micronuclei than in the solvent control, indicative of an apparent threshold. Clear evidence for genotoxic effects was also found in PUBEC cultures treated with OTA concentrations of 1 μM and more, although the dose-effect relationship in PUBEC was more variable for several freshly isolated cell batches, pointing to differences in susceptibility to OTA between bladder cells from different donor animals. The chromosomal genotoxicity of OTA demonstrated in this study is in general accord with previous findings on the induction of clastogenic effects and oxidative DNA damage by OTA. In both cases, the shape of the dose-response curve at very low OTA concentrations supports the existence of a threshold for its genotoxicity.

In vitro study of cell survival following dynamic MLC intensity‐modulated radiation therapy dose deliverya)

The possibility of reduced cell kill following intensity-modulated radiation therapy (IMRT) compared to conventional radiation therapy has been debated in the literature. This potential reduction in cell kill relates to prolonged treatment times typical of IMRT dose delivery and consequently increased repair of sublethal lesions. While there is some theoretical support to this reduction in cell kill published in the literature, direct experimental evidence specific to IMRT dose delivery patterns is lacking. In this study we present cell survival data for three cell lines: Chinese hamster V79 fibroblasts, human cervical carcinoma, SiHa and colon adenocarcinoma, WiDr. Cell survival was obtained for 2.1 Gy delivered as acute dose with parallel-opposed pair (POP), irradiation time 75 s, which served as a reference; regular seven-field IMRT, irradiation time 5 min; and IMRT with a break for multiple leaf collimator (MLC) re-initialization after three fields were delivered, irradiation time 10 min. An actual seven-field dynamic MLC IMRT plan for a head and neck patient was used. The IMRT plan was generated for a Varian EX or iX linear accelerator with 120 leaf Millenium MLC. Survival data were also collected for doses 1X, 2X, 3X, 4X, and 5x 2.1 Gy to establish parameters of the linear-quadratic equation describing survival following acute dose delivery. Cells were irradiated inside an acrylic cylindrical phantom specifically designed for this study. Doses from both IMRT and POP were validated using ion chamber measurements. A reproducible increase in cell survival was observed following IMRT dose delivery. This increase varied from small for V79, with a surviving fraction of 0.8326 following POP vs 0.8420 following uninterrupted IMRT, to very pronounced for SiHa, with a surviving fraction of 0.3903 following POP vs 0.5330 for uninterrupted IMRT. When compared to IMRT or IMRT with a break for MLC initialization, cell survival following acute dose delivery was significantly different, p < 0.05, in three out of six cases. In contrast, when cell survival following IMRT was compared to that following IMRT with a break for MLC initialization the difference was always statistically insignificant. When projected to a 30 fraction treatment, dose deficit to bring cell survival to the same value as in POP was calculated as 4.1, 24.9, and 31.1 Gy for V79, WiDr, and SiHa cell lines, respectively. The dose deficit did not relate to the alpha/beta ratio obtained in this study for the three cell lines. Clinical data do not show reduction in local control following IMRT. Possible reasons for this are discussed. The obtained data set can serve as a test data set for models designed to explore the effect of dose delivery prolongation/fractionation in IMRT on radiation therapy outcome.

Does the cellular labile iron pool participate in the oxidation of 2′,7′-dichlorodihydrofluorescein?

The fluorogenic probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) is widely used for the estimation of oxidative stress in cells. It is known that 2′,7′-dichlorodihydrofluorescein (H2DCF), product of intracellular hydrolysis of H2DCF-DA, is oxidized to the fluorescent compound, DCF, mainly by hydrogen peroxide (H2O2) in the presence of catalysts. The present study was aimed at answering the question whether the labile iron pool (LIP) may contribute to the oxidation of H2DCF in cellular systems. The membrane-permeable lipophilic iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH) was found to inhibit oxidation of the probe by H2O2 dependent on ferrous ions but not by peroxidase or superoxide dismutase in defined in vitro systems. When applied to cells, the probe inhibited considerably oxidation of H2DCF in V79 Chinese hamster fibroblasts and two murine lymphoma L5178Y(LY) sublines (LY-R, LY-S) differing in LIP level, the extent of inhibition being greater in the LY-R line of higher LIP level. These results demonstrate that LIP is a significant factor determining the rate of intracellular H2DCF oxidation.

Toxicological and Analytical Investigations of Noni (Morinda citrifolia) Fruit Juice

Morinda citrifolia (noni) is known to contain genotoxic anthraquinones in the roots. Because of the widespread use of noni juice, the possible genotoxic risk was examined through a battery of short-term tests. Noni juice was also chemically analyzed for the possible presence of anthraquinones. Noni juice extract in the Salmonella microsome assay showed a slight mutagenic effect in strain TA1537, due to the presence of flavonoids. No mutagenicity was observed in the mammalian mutagenicity test with V79 Chinese hamster fibroblasts. Rats treated with a noni juice concentrate did not show DNA repair synthesis (UDS) in primary rat hepatocytes, nor could DNA adducts or DNA strand breaks be observed. HPLC analysis of noni juice for anthraquinones was negative, with a sensitivity of <1 ppm. In summary, chemical analysis and genotoxicity tests reveal that noni juice does not have a genotoxic potential and that genotoxic anthraquinones do not exist in noni juice.

Molecular and functional characterization of microsomal UDP-glucuronic acid uptake by members of the nucleotide sugar transporter (NST) family.

Transport of the co-substrate UDPGA (UDP-glucuronic acid) into the lumen of the endoplasmic reticulum is an essential step in glucuronidation reactions due to the intraluminal location of the catalytic site of the enzyme UGT (UDP-glucuronosyltransferase). In the present study, we have characterized the function of several NSTs (nucleotide sugar transporters) and UGTs as potential carriers of UDPGA for glucuronidation reactions. UDPGlcNAc (UDP-N-acetylglucosamine)-dependent UDPGA uptake was found both in rat liver microsomes and in microsomes prepared from the rat hepatoma cell line H4IIE. The latency of UGT activity in microsomes derived from rat liver and V79 cells expressing UGT1A6 correlated well with mannose-6-phosphatase latency, confirming the UGT in the recombinant cells retained a physiology similar to rat liver microsomes. In the present study, four cDNAs coding for NSTs were obtained; two were previously reported (UGTrel1 and UGTrel7) and two newly identified (huYEA4 and huYEA4S). Localization of NSTs within the human genome sequence revealed that huYEA4S is an alternatively spliced form of huYEA4. All the cloned NSTs were stably expressed in V79 (Chinese hamster fibroblast) cells, and were able to transport UDPGA after preloading of isolated microsomal vesicles with UDPGlcNAc. The highest uptake was seen with UGTrel7, which displayed a V(max) approx. 1% of rat liver microsomes. Treatment of H4IIE cells with beta-naphthoflavone induced UGT protein expression but did not affect the rate of UDPGA uptake. Furthermore, microsomes from UGT1-deficient Gunn rat liver showed UDPGA uptake similar to those from control rats. These data show that NSTs can act as UDPGA transporters for glucuronidation reactions, and indicate that UGTs of the 1A family do not function as UDPGA carriers in microsomes. The cell line H4IIE is a useful model for the study of UDPGA transporters for glucuronidation reactions.

Sci‐Sat AM (1) General‐01: In vitro measurement of cell kill by IMRT fields

Results of the in vitro measurements of cell kill from IMRT fields compared to acute dose delivery are presented. Survival was assayed for three cell lines: Chinese hamster V79 fibroblasts, human cervical carcinoma SiHa and colon adenocarcinoma WiDr. An actual head and neck seven field IMRT plan produced for Varian iX, 120 Millennium MLC linear accelerator was used. The calculated IMRT dose to the point of irradiation in the acrylic phantom was 2.1Gy, verified by measurements with an IC10 ionization chamber. The dose varied within 2% through dimensions of the specimen. Two scenarios were explored: 1. regular IMRT delivery, irradiation time of approximately 5min, and 2. IMRT delivery which required MLC re‐initialization after three fields were delivered, irradiation time of 10min. For comparison the same dose of 2.1Gy was delivered by parallel‐opposed pair (POP) in 75s. Persistent increase in cell survival following irradiation with IMRT fields was observed for all cell lines. This increase varied from small for V79 cells: 0.833±0.018 (95% confidence limits) from POP compared to 0.860±0.040 for IMRT with MLC re‐initialization, to very pronounced for the radiosensitive SiHa cell line: 0.390±0.046 for POP irradiation compared to 0.591±0.080 from IMRT requiring MLC re‐initialization. For WiDr cell line survival was 0.744±0.038 for POP, and 0.809±0.023 for regular IMRT. We observed a persistent, although not always statistically significant increase in cell survival from IMRT fields. While this study does not account for features present in vivo, it provides useful insight into biological consequences of IMRT as assayed by cell survival.

SU‐FF‐T‐460: Comparative in Vitro Study of Cell Survival Following IMRT and Acute Dose Delivery

Purpose: To investigate cell kill from IMRT fields compared to acute dose delivery. Method and Materials: Survival was assayed in vitro for three cell lines: Chinese hamster V79 fibroblasts, human cervical carcinoma SiHa and colon adenocarcinoma WiDr. An actual head and neck seven field dynamic IMRT plan produced for Varian iX, 120 Millennium MLC linear accelerator was used. The calculated IMRT dose (Cadplan, Palo Alto) to the point of irradiation in the acrylic phantom was 2.1Gy. This was verified by measurements with an IC10 ionization chamber. Two IMRT dose delivery scenarios were explored: 1. normal delivery, irradiation time of 5min 10s, and 2. IMRT delivery with a 5min break for MLC re‐initialization after three fields were delivered, irradiation time of 10min. For comparison the same dose of 2.1Gy was delivered by parallel‐opposed pair (POP) in 75s, 20s beam‐on time per beam. Survival data were obtained in the dose range up to 10.5Gy to establish the linear‐quadratic survival curves. Results: An increased cell survival following irradiation with IMRT fields was observed for all cell lines. V79 cells showed the smallest increase: 0.833±0.018 (95% confidence limits) from POP compared to 0.860±0.040 for IMRT with MLC re‐initialization. This increase was very pronounced for the radiosensitive SiHa cell line: 0.390±0.046 for POP irradiation compared to 0.591±0.080 from IMRT requiring MLC re‐initialization. Although variable between cell lines, projections made for a 30 fraction treatment showed substantial reduction in cell kill. Conclusions: We observed an increase in cell survival from IMRT fields compared to acutely delivered dose. This increase was persistent, but not always statistically significant. Projections for a fractionated treatment showed that consequences of this increased cell survival are substantial with a very large variation between cell lines. This projection, however, does not account for features present in vivo, for example reoxygenation and reassortment.

Effect of glutathione depletion on apoptosis induced by thiram in Chinese hamster fibroblasts

Fungicide thiram, which is also known as an inducer of allergic contact dermatitis (ACD), was used as a model compound of thiuram chemicals, and its cellular effects were investigated in cultured Chinese hamster V79 cells. The level of intracellular reduced glutathione (GSH), protein sulfhydryl (PSH) groups, protein carbonyls (PC), membrane lipid peroxidation reflected by enhanced thiobarbituric acid reactive substrates (TBARS) production, as well as apoptotic effect were determined. The apoptosis induction was determined by assessing DNA fragmentation by TUNEL, annexin V binding, and caspases activation assays, using fluorescent microscope or flow cytometry, respectively. The concentrations of thiram required to induce cellular GSH depletion (by 40–50%), protein, and membrane lipid peroxidation (2-fold, and 1.7-fold, respectively), as well as to induce apoptosis in V79 Chinese hamster fibroblasts without causing necrosis through cytotoxic effects were between 50–100 μM. To investigate the role of decreased GSH content in the toxicity of thiram, GSH level was modified prior to exposure. Pretreatment of V79 cells with N-acetyl- l-cysteine (NAC), a GSH biosynthesis precursor, prevented GSH decrease, PC and TBARS production, as well as caspases activation induced by thiram exposure. On the other hand, thiram effects were enhanced by the previous depletion of cellular GSH by l-buthionine-(S,R)-sulfoximine (BSO).

Multiplex Polymerase Chain Reaction Analysis of UV‐A– and UV‐B–induced Delayed and Early Mutations in V79 Chinese Hamster Cells¶

ABSTRACTWe previously reported that approximately 10% of V79 Chinese hamster fibroblast populations clonally derived from single cells immediately after irradiation with either ultraviolet B (UV‐B, 290–320 nm, mainly 311 nm) or ultraviolet A (UV‐A, 320–400 nm, mainly 350–390 nm) radiation exhibit genomic instability. The instability is revealed by relatively high mutation frequencies in the hypoxanthine phosphoribosyl transferase (hprt) gene up to 23 cell generations after irradiation. These delayed mutant clones exhibited higher levels of oxidative stress than normal cells. Therefore, persistently increased oxidative stress has been proposed as a mechanism for UV‐induced genomic instability. This study investigates whether this mechanism is reflected in the deletion spectrum of delayed mutant clones. Eighty‐eight percent of the delayed mutant clones derived from UV‐A–irradiated populations were found to have total deletion of the hprt gene. Correspondingly, 81% of UV‐A–induced early mutations (i.e. detected shortly after irradiation) also had total deletions. Among delayed UV‐B–induced mutant clones, 23% had total deletions and 8% had deletion of one exon, whereas all early UV‐B events were either point mutations or small deletions or insertions. In conclusion, the multiplex polymerase chain reaction deletion screen showed that there were explicit differences in the occurrence of large gene alterations between early and delayed mutations induced by UV‐B radiation. For UV‐A radiation the deletion spectra were similar for delayed and early mutations. UV‐A radiation is, in contrast to UV‐B radiation, only weakly absorbed by DNA and probably induces mutation almost solely via production of reactive oxygen species. Therefore, the present results support the hypothesis that persistent increase in oxidative stress is involved in the mechanism of UV‐induced genomic instability.

Multiplex Polymerase Chain Reaction Analysis of UV-A– and UV-B–induced Delayed and Early Mutations in V79 Chinese Hamster Cells¶

We previously reported that approximately 10% of V79 Chinese hamster fibroblast populations clonally derived from single cells immediately after irradiation with either ultraviolet B (UV-B, 290-320 nm, mainly 311 nm) or ultraviolet A (UV-A, 320-400 nm, mainly 350-390 nm) radiation exhibit genomic instability. The instability is revealed by relatively high mutation frequencies in the hypoxanthine phosphoribosyl transferase (hprt) gene up to 23 cell generations after irradiation. These delayed mutant clones exhibited higher levels of oxidative stress than normal cells. Therefore, persistently increased oxidative stress has been proposed as a mechanism for UV-induced genomic instability. This study investigates whether this mechanism is reflected in the deletion spectrum of delayed mutant clones. Eighty-eight percent of the delayed mutant clones derived from UV-A-irradiated populations were found to have total deletion of the hprt gene. Correspondingly, 81% of UV-A-induced early mutations (i.e. detected shortly after irradiation) also had total deletions. Among delayed UV-B-induced mutant clones, 23% had total deletions and 8% had deletion of one exon, whereas all early UV-B events were either point mutations or small deletions or insertions. In conclusion, the multiplex polymerase chain reaction deletion screen showed that there were explicit differences in the occurrence of large gene alterations between early and delayed mutations induced by UV-B radiation. For UV-A radiation the deletion spectra were similar for delayed and early mutations. UV-A radiation is, in contrast to UV-B radiation, only weakly absorbed by DNA and probably induces mutation almost solely via production of reactive oxygen species. Therefore, the present results support the hypothesis that persistent increase in oxidative stress is involved in the mechanism of UV-induced genomic instability.

Disturbed microtubule function and induction of micronuclei by chelate complexes of mercury(II)

Interactions of mercury(II) with the microtubule network of cells may lead to genotoxicity. Complexation of mercury(II) with EDTA is currently being discussed for its employment in detoxification processes of polluted sites. This prompted us to re-evaluate the effects of such complexing agents on certain aspects of mercury toxicity, by examining the influences of mercury(II) complexes on tubulin assembly and kinesin-driven motility of microtubules. The genotoxic effects were studied using the micronucleus assay in V79 Chinese hamster fibroblasts. Mercury(II) complexes with EDTA and related chelators interfered dose-dependently with tubulin assembly and microtubule motility in vitro. The no-effect-concentration for assembly inhibition was 1 μM of complexed Hg(II), and for inhibition of motility it was 0.05 μM, respectively. These findings are supported on the genotoxicity level by the results of the micronucleus assay, with micronuclei being induced dose-dependently starting at concentrations of about 0.05 μM of complexed Hg(II). Generally, the no-effect-concentrations for complexed mercury(II) found in the cell-free systems and in cellular assays (including the micronucleus test) were identical with or similar to results for mercury tested in the absence of chelators. This indicates that mercury(II) has a much higher affinity to sulfhydryls of cytoskeletal proteins than to this type of complexing agents. Therefore, the suitability of EDTA and related compounds for remediation of environmental mercury contamination or for other detoxification purposes involving mercury has to be questioned.

Aluminium triggers necrosis and apoptosis in V79 cells

The aim of this work was to examine the induction of cell death by aluminium (Al) at low pH, where the toxic cation Al3+ has maximum activity. Chinese hamster V79 fibroblasts were used as a model to study the toxic effects of different Al concentrations under optimal conditions for Al3+ activity (pH 4.5). Al3+ was detected by 8-hydroxyquinoline staining and cell death was assessed by image analysis, TUNEL assay and annexin-V labelling. Intracellular Al3+ deposits occurred mainly in the cytoplasm and within the nucleus. Al3+ altered the nuclear morphology, with chromatin condensation and loss being observed in Al3+-treated cells. Necrosis was detected with 10−5–10−3 M AlCl3 and apoptosis was observed only at 10−8–10−5 M AlCl3. Our results show that Al3+ induces cell death through a mechanism which could determine whether necrosis or apoptosis would be triggered.

Enhancement of catalase activity by repetitive low-grade H 2O 2 exposures protects fibroblasts from subsequent stress-induced apoptosis

Exposure of Chinese hamster V79 fibroblasts to mild and repetitive H 2O 2 doses in culture for 15 weeks produced no change in lipid peroxidation status, GSH/GSSG ratio and glutathione peroxidase activity of these cells (VST cells). In contrast, in VST cells catalase levels underwent a prominent increase which could be significantly inhibited and brought down to control levels after treatment with the catalase inhibitor 3-aminotriazole (3-AT). When control (VC) cells were exposed to UV radiation (UVC 5 J/m 2) or H 2O 2 (7.5 mM, 15 min), intracellular reactive oxygen species (ROS) levels rose prominently with significant activation of caspase-3. Marked nuclear fragmentation and lower cell viability were also noted in these cells. In contrast, VST cells demonstrated a significantly lower ROS level, an absence of nuclear fragmentation and an unchanged caspase-3 activity after exposure to UVC or H 2O 2. Cell viability was also significantly better preserved in VST cells than VC cells after UV or H 2O 2 exposures. Following 3-AT treatment of VST cells, UVC radiation or H 2O 2 brought about significantly higher elevations in intracellular ROS, increases in caspase-3 activity, significantly lowered cell viability and marked nuclear fragmentation, indicating the involvement of high catalase levels in the cytoprotective effects of repetitive stress. Therefore, upregulation of the antioxidant defense after repetitive oxidative stress imparted a superior ability to cope with subsequent acute stress and escape apoptotic death and loss of viability.

Interaction of metal salts with cytoskeletal motor protein systems

Interactions of chemicals with the microtubular network of cells may lead to genotoxicity. Micronuclei (MN) might be caused by interaction of metals with tubulin and/or kinesin. The genotoxic effects of inorganic lead and mercury salts were studied using the MN assay and the CREST analysis in V79 Chinese hamster fibroblasts. Effects on the functional activity of motor protein systems were examined by measurement of tubulin assembly and kinesin-driven motility. Lead and mercury salts induced MN dose-dependently. The no-effect-concentration for MN induction was 1.1 μM PbCl 2, 0.05 μM Pb(OAc) 2 and 0.01 μM HgCl 2. The in vitro results obtained for PbCl 2 correspond to reported MN induction in workers occupationally exposed to lead, starting at 1.2 μM Hg(II) (Vaglenov et al., 2001, Environ. Health Perspect. 109, 295–298). The CREST Analysis indicate aneugenic effects of Pb(II) and aneugenic and additionally clastogenic effects of Hg(II). Lead (chloride, acetate, and nitrate) and mercury (chloride and nitrate) interfered dose-dependently with tubulin assembly in vitro. The no-effect-concentration for lead salts in this assay was 10 μM. Inhibition of tubulin assembly by mercury started at 2 μM. The gliding velocity of microtubules along immobilised kinesin molecules was affected by 25 μM Pb(NO 3) 2 and 0.1 μM HgCl 2 in a dose-dependent manner. Our data support the hypothesis that lead and mercury genotoxicity may result, at least in part, via disturbance of chromosome segregation via interaction with cytoskeletal proteins.