V79 Chinese Hamster Cell Line Research Articles

Monte Carlo simulations of cell survival in proton SOBP.

Objective. The objective of this study is to develop a multi-scale modeling approach that accurately predicts radiation-induced DNA damage and survival fraction in specific cell lines.Approach. A Monte Carlo based simulation framework was employed to make the predictions. The FLUKA Monte Carlo code was utilized to estimate absorbed doses and fluence energy spectra, which were then used in the Monte Carlo Damage Simulation code to compute DNA damage yields in Chinese hamster V79 cell lines. The outputs were converted into cell survival fractions using a previously published theoretical model. To reduce the uncertainties of the predictions, new values for the parameters of the theoretical model were computed, expanding the database of experimental points considered in the previous estimation. Simulated results were validated against experimental data, confirming the applicability of the framework for proton beams up to 230 MeV. Additionally, the impact of secondary particles on cell survival was estimated.Main results. The simulated survival fraction versus depth in a glycerol phantom is reported for eighteen different configurations. Two proton spread out Bragg peaks at several doses were simulated and compared with experimental data. In all cases, the simulations follow the experimental trends, demonstrating the accuracy of the predictions up to 230 MeV.Significance. This study holds significant importance as it contributes to the advancement of models for predicting biological responses to radiation, ultimately contributing to more effective cancer treatment in proton therapy.

Prediction of DNA rejoining kinetics and cell survival after proton irradiation for V79 cells using Geant4-DNA

Purpose:Track structure Monte Carlo (MC) codes have achieved successful outcomes in the quantitative investigation of radiation-induced initial DNA damage. The aim of the present study is to extend a Geant4-DNA radiobiological application by incorporating a feature allowing for the prediction of DNA rejoining kinetics and corresponding cell surviving fraction along time after irradiation, for a Chinese hamster V79 cell line, which is one of the most popular and widely investigated cell lines in radiobiology. Methods:We implemented the Two-Lesion Kinetics (TLK) model, originally proposed by Stewart, which allows for simulations to calculate residual DNA damage and surviving fraction along time via the number of initial DNA damage and its complexity as inputs. Results:By optimizing the model parameters of the TLK model in accordance to the experimental data on V79, we were able to predict both DNA rejoining kinetics at low linear energy transfers (LET) and cell surviving fraction. Conclusion:This is the first study to demonstrate the implementation of both the cell surviving fraction and the DNA rejoining kinetics with the estimated initial DNA damage, in a realistic cell geometrical model simulated by full track structure MC simulations at DNA level and for various LET. These simulation and model make the link between mechanistic physical/chemical damage processes and these two specific biological endpoints.

Radiation-Induced Mutagenesis in Mammalian Cells after Exposure to Accelerated Ions with Different LET

The mutagenic effect of accelerated heavy charged particles on the Chinese hamster V79 cell line is studied. The induction of HPRT mutations in irradiated cells for a long expression time (up to 45 days) after exposure to radiation with different LET are analyzed. The maximum yield of mutant clones is observed at different expression times, depending on the characteristics of ionizing radiation; in this case, the position of the maximum is shifted toward later time intervals with increasing LET of radiation.

In vitro evaluation of the cytotoxicity of chlorhexidine digluconate, povidone iodine and phenolic compound mouth washes on chinese hamster v79 cell lines

Aim: To assess and compare the cytotoxic potential of Chlorhexidine, Listerine and Povidone –Iodine antimicrobial agents on Chinese hamster lung fibroblast V79 cells. Materials and Method: Chlorhexidine (0.2%), Listerine, Povidone iodine were diluted with DMEM medium under sterile conditions to obtain the required concentration. V79 cells were used for the experimental procedures. The cells were cultured in DMEM media supplemented with 10% FBS and 1% Gentamycin at 370 C in a humidified 5% CO2 incubator. For all the assays, cells were maintained at 80-90% confluency in T-25 cm2 flasks and used as per requirement. V79 cells were seeded onto 96 well plates at a density of 104 cells per well and allowed to incubate overnight at 37 0 C in 5% CO2 incubator. The following day the cells were treated with different concentrations of Betadine, Hexidine and Listerine for 12, 24 and 48 hours. Following the treatments, the drug containing media was discarded and 100µl of freshly prepared MTT stock prepared in DMEM media was added in to each well and the plates were incubated at 370 C in 5% CO2 incubator for 4 hours. Viability was determined by the ability of the cells to reduce MTT, a water soluble tetrazolium dye to produce violet crystals of formazan. Formazan crystals formed were solubilized by the addition of 100µl of (Di Methyl Sulfoxide). Optical density which is directly proportional to cell viability, was measured at 540nm using a multi well spectrophotometer. Viability curves were plotted using origin. Percentage cell viability was obtained using the formula given below: Percentage viability= [(OD Test – OD Blank)/ (OD Control – OD Blank)] *100 Results: V79 cells treated with serially diluted test compounds showed a decrease in cellular viability in a concentration as well as time dependent manner. Hexidine exhibited maximum toxic effect when compared to Listerine and Betadine; betadine possessing the least toxic nature as obvious by the IC50 values for different time durations. Conclusion: Chlorhexidine was found to be most toxic when compared to Listerine and Povidone iodine. Povidone iodine being the least toxic amongst the three mouth rinse. All the compounds exhibited toxic effect in a dose dependent as well as time dependent manner. The result of this study showed that all three given mouth rinses caused V79 proliferation inhibition.

Induction of micronuclei and cell cycle arrest by some tri- and tetrachlorobiphenyls in mammalian cells deficient in xenobiotic-metabolizing enzymes.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants with continued public health concerns. The lower chlorinated biphenyls are supposed to be mutagenic following metabolic activation. However, in a preliminary study, we recently observed induction of micronuclei by several PCBs in a subclone of Chinese hamster V79 cell line, V79-Mz, which is deficient in xenobiotic-metabolizing enzyme activities. In this study, metabolism-free genotoxicity of PCBs was investigated, using 10 tri- and tetrachlorobiphenyls, in V79, V79-Mz, and human hepatoma (HepG2) cell lines. Among the four tetrachlorobiphenyls, 2,4,4',5- and 2,3'4,4'-tetrachlorobiphenyl-both having a noncoplanar configuration-induced micronuclei in V79-Mz cells, while their coplanar analogs 3,4,4',5- and 3,3',4,4'-tetrachlorobiphenyl were inactive. Furthermore, 2,3,3'- (PCB 20) and 2,3,4'-trichlorobiphenyl (PCB 22) started to induce micronuclei in V79-Mz cells at 10 μM and higher concentrations, demonstrating more potent effects than observed with 2,2',3-, 2,2',4-, 2,2',5, and 2,4,4'-trichlorobiphenyl. As representative compounds, PCB 20 and 22 induced micronuclei in relatively high concentrations in HepG2 cells (p53-proficient), though they did not induce Hprt gene mutations in V79-Mz cells. PCB 20 and 22 increased mitotic index and induced cell cycle arrest at the G2/M phase, with effects more potent in V79-Mz than in V79 cells. This study suggests that 2,3,4'- and 2,3,3'-substituted PCBs are micronuclei inducers and G2/M arresters among a number of trichlorobiphenyls in mammalian cell lines, though with potency lower than that observed recently in V79-derived cells expressing human CYP2E1. Similarly, some noncoplanar tetrachlorobiphenyls possess metabolism-independent chromosome-damaging potentials. Environ. Mol. Mutagen. 58:199-208, 2017. © 2017 Wiley Periodicals, Inc.

Antigenotoxic, anti-photogenotoxic and antioxidant activities of natural naphthoquinone shikonin and acetylshikonin and Arnebia euchroma callus extracts evaluated by the umu-test and EPR method

The aim of this study was to evaluate the antigenotoxic and antioxidant potential of shikonin (SH), acetylshikonin (ACS) and Arnebia euchroma callus extract (EXT). The antigenotoxic activity was investigated by the umu-test as the inhibition of the SOS system induction caused by genotoxic chemical agents — 4-nitroquinoline oxide and 2-aminoanthracene. Moreover the ability of SH, ACS and EXT to prevent photogenotoxicity triggered by chlorpromazine under UVA irradiation was measured. The cytotoxicity of EXT toward V79 Chinese hamster cell line was additionally assessed. Shikonin and acetylshikonin had no effect on 4-NQO induced genotoxicity whereas EXT demonstrated an unclear effect. The protection against 2AA induced genotoxicity was observed for all tested substances. The highest protection was demonstrated for EXT with inhibition of 66%. SH and ACS reduced 2AA genotoxicity with inhibition of about 60%. Under UVA the strongest and dose-dependent activity was observed for EXT. Acetylshikonin was a weak anti-photogenotoxin whereas shikonin had no clear effect. EXT was highly cytotoxic toward the V79 cell line — the cells' morphology was affected seriously and apoptosis was impacted.The antioxidant activity of SH, ACS and EXT was studied by means of electron paramagnetic resonance spectroscopy using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. All three samples exhibited radical scavenging properties.

Evaluation of photodegradation, phototoxicity and photogenotoxicity of ofloxacin in ointments with sunscreens and in solutions

Fluoroquinolones are widely used anti-bacterial agents that are known to exhibit moderate to severe phototoxicity. Furthermore some of them reveal photogenotoxicity under UV irradiation. Incidence of side effects due to light exposure may be augmented, if the medicament is used topically. The main goal of this work was to compare the extent of photodegradation of ofloxacin in ointments with various excipients: hydrated or non-hydrated base and the addition of sunscreens: bisoctrizole (Tinosorb® M) and bemotrizinol (Tinosorb® S). The next goal of present work was the analysis of phototoxicity and photogenotoxicity of ofloxacin photodegradation products in tested ointments and in solutions with the umu-test, the test of mitotic gene conversion with Saccharomyces cerevisiae D7 and the micronucleus assay with V79 Chinese hamster cell line. At the same time an attempt was made to determinate the photodegradation products of ofloxacin in different unguents variants. We observed a significant photoprotective effect in ointment with Tinosorb® M. We did not evaluated relevant differences regarding the genotoxicity and toxicity of unguents. However, the pre-irradiated ofloxacin solutions in comparison to samples stored in the dark were significantly more genotoxic to bacteria, slightly increased the number of micronuclei in V79 cell line and were toxic to the yeast strain.

Ruthenium(II)/4,6-dimethyl-2-mercaptopyrimidine complexes: Synthesis, characterization, X-ray structures and in vitro cytotoxicity activities on cancer cell lines

The complexes [RuCl(SpymMe2-N,S)2(NO)] (1) and [Ru(dppb)(SpymMe2-N,S)2] (2) (dppb=1,4-bis(diphenylphosphino)butane, SpymMe2=deprotonated 4,6-dimethyl-2-mercaptopyrimidine) were obtained and characterized by elemental analysis, spectroscopic techniques including X-ray crystallography, conductimetry and cyclic voltammetry. Energy Decomposition Analysis (EDA) calculation indicated strong intermolecular interaction between monomers of complex 1, as suggested by the 1H NMR spectrum of this complex, in chloroform solution. Additionally, preliminary tests were carried out, in order to evaluate the cytotoxicity activity on cancer cell lines of the new complexes, and the free ligands, against MDA-MB-231 (breast cancer), Hela (cervical cancer), U251 (glioma cancer) tumor cell lines and the V79 (Chinese lung fibroblast hamster) cell line. The best result was obtained for the complex 1 with IC50 value of 0.11±0.02, against MDA-MB-231 tumor cells.

Sustained induction of cytochrome P4501A1 in human hepatoma cells by co-exposure to benzo[a]pyrene and 7H-dibenzo[c,g]carbazole underlies the synergistic effects on DNA adduct formation

To gain a deeper insight into the potential interactions between individual aromatic hydrocarbons in a mixture, several benzo[a]pyrene (B[a]P) and 7H-dibenzo[c,g]carbazole (DBC) binary mixtures were studied. The biological activity of the binary mixtures was investigated in the HepG2 and WB-F344 liver cell lines and the Chinese hamster V79 cell line that stably expresses the human cytochrome P4501A1 (hCYP1A1). In the V79 cells, binary mixtures, in contrast to individual carcinogens, caused a significant decrease in the levels of micronuclei, DNA adducts and gene mutations, but not in cell survival. Similarly, a lower frequency of micronuclei and levels of DNA adducts were found in rat liver WB-F344 cells treated with a binary mixture, regardless of the exposure time. The observed antagonism between B[a]P and DBC may be due to an inhibition of Cyp1a1 expression because cells exposed to B[a]P:DBC showed a decrease in Cyp1a1 mRNA levels. In human liver HepG2 cells exposed to binary mixtures for 2h, a reduction in micronuclei frequency was also found. However, after a 24h treatment, synergism between B[a]P and DBC was determined based on DNA adduct formation. Accordingly, the up-regulation of CYP1A1 expression was detected in HepG2 cells exposed to B[a]P:DBC. Our results show significant differences in the response of human and rat cells to B[a]P:DBC mixtures and stress the need to use multiple experimental systems when evaluating the potential risk of environmental pollutants. Our data also indicate that an increased expression of CYP1A1 results in a synergistic effect of B[a]P and DBC in human cells. As humans are exposed to a plethora of noxious chemicals, our results have important implications for human carcinogenesis.

Does formaldehyde induce aneuploidy?

Formaldehyde (FA) was tested for a potential aneugenic activity in mammalian cells. We employed tests to discriminate between aneugenic and clastogenic effects in accordance with international guidelines for genotoxicity testing. The cytokinesis-block micronucleus test (CBMNT) in combination with fluorescence in situ hybridisation (FISH) with a pan-centromeric probe was performed with cultured human lymphocytes and the human A549 lung cell line. FA induced micronuclei (MN) in binuclear cells of both cell types under standard in vitro test conditions following the OECD guideline 487. FISH analysis revealed that the vast majority of induced MN were centromere negative, thus indicating a clastogenic effect. A similar result was obtained for MN induced by γ-irradiation, whereas the typical aneugens colcemid (COL) and vincristine (VCR) predominantly induced centromere-positive MN. Furthermore, COL and VCR clearly enhanced the MN frequency in mononuclear lymphocytes in the CBMNT, whereas such an effect was not observed for γ-irradiation and FA. In experiments with the Chinese hamster V79 cell line, the aneugens COL and VCR clearly increased the frequency of tetraploid second division metaphases, whereas FA did not cause such an effect. Altogether, our results confirm the clastogenicity of FA in cultured mammalian cells but exclude a significant aneugenic activity.

5-Fluorouracil, colchicine, benzo[a]pyrene and cytosine arabinoside tested in the in vitro mammalian cell micronucleus test (MNvit) in Chinese hamster V79 cells at Covance Laboratories, Harrogate, UK in support of OECD draft Test Guideline 487

The reference genotoxic agents 5-fluorouracil (a nucleoside analogue, characterised by a steep dose response profile), colchicine (an aneugen that inhibits tubulin polymerisation), benzo[a]pyrene (a polycyclic aromatic hydrocarbon requiring metabolic activation) and cytosine arabinoside (a nucleoside analogue that inhibits the gap-filling step of excision repair) were tested in the in vitro micronucleus assay using the Chinese hamster V79 cell line at Covance Laboratories, Harrogate, UK. All chemicals were treated in the absence and presence of cytokinesis block (via addition of cytochalasin B) with this work forming part of a collaborative evaluation of the toxicity measures recommended in the draft OECD Test Guideline 487 on the In Vitro Mammalian Cell Micronucleus Test (MNvit). The toxicity measures used, detecting a possible combination of both cytostasis and cell death (though not cell death directly), were relative population doubling, relative increase in cell counts and relative cell counts for treatments in the absence of cytokinesis block, and replication index in the presence of cytokinesis block. All of the chemicals tested either gave marked increases in the percentage of micronucleated cells with and without cytokinesis block, or did not induce micronuclei at concentrations giving approximately 50–60% toxicity (cytostasis and cell death) or less by all of the toxicity measures used. The outcome from this series of tests supports the use of relative increase in cell counts and relative population doubling, as well as relative cell counts, as appropriate measures of cytotoxicity for the non-cytokinesis blocked in vitro micronucleus assay.

Mutator phenotype of mammalian cells due to deficiency of NEIL1 DNA glycosylase, an oxidized base-specific repair enzyme

The recently characterized NEIL1 and NEIL2 are distinct from the previously characterized mammalian DNA glycosylases (OGG1 and NTH1) involved in repair of oxidized bases because of the NEILs’ preference for excising base lesions from single-stranded DNA present in bubble and fork structures. OGG1 and NTH1 are active only with duplex DNA. This raises the possibility that NEILs function in the repair of base lesions during DNA replication and/or transcription. S-phase-specific activation of only NEIL1 suggests its preferential involvement in repair during DNA replication. Here we show that antisense oligonucleotides specific for human or Chinese hamster NEIL1 decreased in vivo NEIL1 levels by 70–80%, concomitant with increased oxidative damage in the genome. Moreover, NEIL1 downregulation enhanced spontaneous mutation in the Hprt locus by about 3-fold in both Chinese hamster V79 and human bronchial A549 cell lines. The mutant frequency was further enhanced (7–8-fold) under oxidative stress. The majority of both spontaneous and induced mutations occurred at A·T base pairs, indicating that oxidized A and/or T are NEIL1's preferred in vivo substrates. NEIL1 thus plays a distinct and important role in repairing endogenous and induced mutagenic oxidized bases, and hence in maintaining the functional integrity of mammalian genomes.

Mechanistic investigations of low dose exposures to the genotoxic compounds bisphenol-A and rotenone

A complete hazard and risk assessment of any known genotoxin requires the evaluation of the mutagenic, clastogenic and aneugenic potential of the compound. In the case of aneugenic chemicals, mechanism of action (MOA) and quantitative responses may be investigated by studying their effects upon the fidelity of functioning of components of the cell cycle. These present studies have demonstrated that the plastics component bisphenol-A (BPA) and the natural pesticide rotenone induce micronuclei and modify the functioning of the microtubule organising centres (MTOCs) of the mitotic spindles of cultured mammalian cells in a dose-dependent manner. BPA and rotenone were used as model compounds in an investigation of dose response relationships for the hazard/risk assessment of aneugens. Thresholds of action for the induction of aneuploidy have been predicted for spindle poisons on the basis of the multiple targets, which may need disabling before a quantitative response can be detected. The cytokinesis blocked micronucleus assay (CBMA) methodology was utilised in the human lymphoblastoid cell lines AHH-1, MCL-5 and Chinese hamster V79 cell lines. A no observable effect level (NOEL) at 10.8 μg/ml BPA was observed for MN induction. Rotenone showed a small increase in MN induction with the first significant effect at 0.25 ng/ml in V79 cells but there was no significant effect in the metabolically competent cell line, MCL-5. For a mechanistic evaluation of the aneugenic effects of BPA and rotenone, fluorescently labelled antibodies were used to visualise microtubules (α-tubulin) and MTOCs (γ-tubulin). The NOELs for tripolar mitotic spindle induction in V79 cells were 7 μg/ml for BPA and 80 pg/ml for rotenone (concentrations which produced similar changes to mitotic index (M.I.)). Interestingly there was close proximity to the NOEL of 10.8 μg/ml BPA for micronucleus (MN) induction in the human lymphoblastoid AHH-1 cell. Multiple MTOCs can therefore be predicted as a possible mechanism for MN induction. The similarity in concentration inducing tripolar mitosis, M.I. and MN changes suggests immunofluorescence analysis to be a useful dose setting assay with emphasis on the mechanism.

Characterization of the genotoxic potential of formaldehyde in V79 cells

Formaldehyde (FA) is known to be genotoxic and mutagenic in proliferating mammalian cells in vitro. The present study was performed to further characterize its genotoxic potential in the V79 Chinese hamster cell line. The induction of DNA strand breaks and DNA-protein cross-links (DPXs) was measured by the comet assay in relationship to the induction of sister chromatid exchanges (SCEs) and micronuclei (MN). Induction of DNA strand breaks was found neither with the standard protocol of the alkaline comet assay nor with modifications using extended electrophoresis times or proteinase K. The concentration-effect relationship for the genotoxic effects was characterized by fitting different curves to the data. A two-phase regression model fitted best in comparison with a linear or a quadratic model and indicated practical thresholds for the induction of SCE and MN. For the induction of DPX as measured by the comet assay, neither a linear concentration-response relationship nor any of the tested models fitted well to the data. Three repeated treatments with genotoxic concentrations of FA with a 3-h interval led to enhanced levels of DPX and MN while the same treatments with a 24-h interval did not enhance FA genotoxicity but suggested adaptive protection against the DNA-damaging action of FA.

Mouse aldo-keto reductase AKR7A5 protects V79 cells against 4-hydroxynonenal-induced apoptosis

We have developed transgenic Chinese hamster V79 cell lines in order to examine the potential for a mouse aldo-keto reductase, AKR7A5, to protect against the toxicity of 4-hydroxynonenal (4-HNE) and related toxic aldehydes. Stable expression of mouse AKR7A5 in V79 cells conferred four-fold increased resistance to 4-HNE cytotoxicity using the MTT assay compared to empty vector-transfected V79 cells. Cells expressing AKR7A5 showed a decrease in mutation rate compared to control cells in the presence of 4-HNE as measured by HGPRT mutagenicity assay. Furthermore, the cells expressing AKR7A5 showed decreased 4-HNE-induced caspase-3 activity in both a time and dose-dependent manner compared to control cells. These results show that in V79 cells 4-HNE mediates apoptosis via caspase-3 activation and that the AKR7A5 enzyme is able to metabolize 4-HNE in cells, thereby attenuating 4-HNE-induced apoptosis. AKR7A isozymes may therefore be important in protecting against toxic aldehydes derived from lipid peroxidation in vivo.

Modulation of radiation response by inhibiting topoisomerase II catalytic activity

Due to the essential role played by DNA topoisomerases (topos) in cell survival, the use of topoisomerase inhibitors as chemotherapeutic drugs in combination with radiation has become a common strategy for the treatment of cancer. Catalytic inhibitors of these enzymes would be promising to improve the effectiveness of radiation and therefore, it appears reasonable to incorporate them in combined modality trials. In this work, we have investigated the capacity of both ICRF-193 and Aclarubicin (ACLA), two catalytic inhibitors of topoisomerase II (Topo II), to modulate radiation response in Chinese hamster V79 cell line and its radiosensitive mutant irs2. We also have explored potential mechanisms underlying these interactions. Experiments were performed in the presence and absence of either ICRF-193 or ACLA, and topo II activity was measured using an assay based upon decatenation of kinetoplast DNA (kDNA). For the combined experiments cells were incubated for 3 h in the presence of various inhibitor concentrations and irradiated 30 min prior to the end of treatments and cell survival was determined by clonogenic assay. DNA-damaging activity was measured by single-cell gel electrophoresis. Our results demonstrate that combinations of catalytic inhibitors of topo II and radiation produce an increase in cell killing induced by ionising radiation. The mechanism of radiation enhancement may involve a direct or indirect participation of topo II in the repair of radiation-induced DNA damage.

Induced resistance in cells exposed to repeated low doses of HO involves enhanced activity of antioxidant enzymes

We have derived cells from the Chinese hamster V79 cell line by conditioning them with repeated low doses of hydrogen peroxide (H(2)O(2)). This mimics the physiological condition where cells are repeatedly exposed to low levels of oxidants. In an attempt to characterize such cells, we have exposed both conditioned cells (V79(C)) and the parental V79 cells (V79(P)) to different types of cytotoxic agents and compared their sensitivity to cell killing. The V79(C) cells were found to be stably resistant to killing by agents that produced toxicity through oxidative stress, e.g. H(2)O(2) and cisplatin. It was also found that the lipid peroxidation produced by these agents were considerably lower in the V79(C) cells. Thus, the difference in sensitivity could be due to lesser extent of damage to these cells. V79(C) cells had greater antioxidant defense through higher GSH content and greater activity of enzymes such as Cu-Zn superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), which provided protection from damage. Enzyme activities were also assayed at different times after treatment with various cytotoxic agents; there was a relatively large increase in SOD activity which perhaps plays a key role in determining the resistance of the V79(C) cells to killing.

DNA adduct formation by 7H‐dibenzo[c,g]carbazole and its tissue‐ and organ‐specific derivatives in Chinese hamster V79 cell lines stably expressing cytochrome P450 enzymes

The cytochrome P4501A subfamily (CYP1A) is involved in the metabolic activation of 7H-dibenzo[c,g]carbazole (DBC) and its tissue- and organ-specific derivatives, N-methyldibenzo[c,g]carbazole (MeDBC)and 5,9-dimethyldibenzo[c,g]carbazole (diMeDBC). In this study, we have evaluated the relationship between the tissue specificity and (32)P-postlabeled adduct patterns produced by these compounds by using a panel of Chinese hamster V79 cell lines stably expressing human CYP1A1 and CYP1A2 and/or N-acetyltransferase. Treatment of the parental cell lines V79MZ and V79NH, which are devoid of any CYP activity, with DBC and its derivatives did not result in detectable adducts. The highest DNA adduct levels were found in CYP1A1-expressing V79MZh1A1 cells after DBC and MeDBC treatment (24.5 +/- 7.2 and 16.2 +/- 3.6 adducts/10(8) nucleotides, respectively). Exposure of this cell line to DBC resulted in five distinct spots, while six spots with different chromatographic mobilities were detected in MeDBC-treated cells. DiMeDBC produced only very low levels of DNA adducts in V79MZh1A1 cells. DBC and MeDBC formed relatively low levels of DNA adducts in CYP1A2-expressing V79MZh1A2 cells (0.7 +/- 0.2 and 2.1 +/- 1.2 adducts/10(8) nucleotides, respectively). DBC formed three weak spots and MeDBC five spots in V79MZh1A2 cells, and all the spots had different chromatographic mobilities. In contrast, diMeDBC did not induce any DNA adducts in these cells, although diMeDBC induced a significant dose-dependent increase in micronucleus frequency under similar treatment conditions (r = 0.76; P < 0.001). The significant increase in DNA damage in the Comet assay following incubation of exposed cells with a repair-specific endonuclease (Fpg protein) suggests that base modifications such as 8-oxodG or Fapy-adducts might be responsible for the genotoxicity of diMeDBC in V79MZh1A2 cells. The similarities between the DNA adduct patterns produced by DBC and MeDBC in V79MZh1A1 and V79MZh1A2 cells suggest that biotransformation mediated via CYP1A1 and CYP1A2 might depend on a PAH-type pathway involving the aromatic ring system.

DNA adduct formation by the environmental contaminant 3-nitrobenzanthrone in V79 cells expressing human cytochrome P450 enzymes.

Diesel exhaust is known to induce tumours in animals. Of the compounds found in diesel exhaust 3-nitrobenzanthrone (3-NBA) is particularly a powerful mutagen. Recently we showed that 3-NBA is genotoxic in vivo in rats by forming specific DNA adducts derived from nitroreduction. In this study a panel of genetically engineered V79 Chinese hamster cell lines expressing various human cytochrome P450 (CYP) enzymes (CYP1A1, CYP3A4) and/or human NADPH:CYP oxidoreductase (CYPOR) was used to identify CYP enzymes involved in the metabolic activation of 3-NBA. We analyzed the formation of specific DNA adducts by 32P-postlabelling after exposing cells to 1 microM 3-NBA. A similar pattern with a total of four distinct 3-NBA-DNA adducts was found in all cells, identical to those detected previously in DNA from rats treated with 3-NBA in vivo. Total adduct levels ranged from 75 to 132 using nuclease P1 and from 103 to 220 adducts per 10(8) nucleotides, using butanol enrichment. Comparison of DNA binding between different V79MZ derived cells revealed that human CYPOR and CYP3A4 were involved in the metabolic activation of 3-NBA. Furthermore, dose-dependent high adduct levels were detected after exposure to 0.01, 0.1 or 1 microM 3-NBA in the subclone V79NH which exhibits high activities of nitroreductase and N,O-acetyltransferase. Our results suggest that nitroreduction is the major pathway in the human bioactivation of 3-NBA. Moreover, acetylation of the initially formed N-hydroxy arylamine intermediates may contribute to the high genotoxic potential of 3-NBA.

Heterologous Expression of Mouse Cytochrome P450 2e1 in V79 Cells: Construction and Characterisation of the Cell Line and Comparison with V79 Cell Lines Stably Expressing Rat P450 2E1 and Human P450 2E1

A V79 Chinese hamster cell line was constructed for stable expression of mouse cytochrome P450 2e1 (Cyp2e1), as an addition to the existing cell battery consisting of cell lines stably expressing rat CYP2E1 and human CYP2E1 (V79 Cell Battery). The aim was to establish a cell battery that offers the in vitro possibility of investigating species-specific differences in the toxicity and metabolism of chemicals representing substrates for CYP2E1. The newly established cell line (V79m2E1) effectively expressed Cyp2e1 in the catalytically active form. The expression of catalytically active CYP2E1 in V79m2E1 cells was maintained over several months in culture, as demonstrated by Western Blotting and chlorzoxazone (CLX) 6-hydroxylase activity. The cells exhibited CLX 6-hydroxylase activity with a Km of 27.8 microM/l and Vmax of 40 pmol/mg protein/minute, compared with a Km of 28.2/28.6 microM/l and a Vmax of 130/60 pmol/mg protein/minute from V79r2E1/V79h2E1 cells. Furthermore, the CYP2E1-dependent mutagenicity of N-nitrosodimethylamine could be demonstrated in the V79m2E1 cells. Therefore, the new cell battery permits the interspecies comparison of CYP2E1-dependent toxicity and of metabolism of chemicals between humans and the two major rodent species--the rat and the mouse--that are usually used in classical toxicity studies.