A simple and sensitive pulsed field gel electrophoresis protocol to study 50 kb apoptotic DNA fragmentation in human lymphocytes

DNA fragmentation of 50 kb is observed in apoptotic human lymphocytes as measured with pulsed field gel electrophoresis (PFGE). Standard PFGE assay involves embedding of cells into agarose blocks followed by lysis in the presence of proteinase K. In this study, we modified the PFGE protocol by omitting the proteinase K. In this study, we modified the PFGE assay by omitting the proteinase K and changing lysis solution according to the method of anomalous viscosity time dependence (AVTD). The conditions of PFGE were adjusted aiming to compress apoptotic fragments, increasing sensitivity and the number of samples that could be loaded on the same gel. Lymphocytes were irradiated with gamma-rays and apoptotic fragmentation of DNA was determined by PFGE using standard lysis with proteinase K and lysis protocol from AVTD method. Both protocols of lysis resulted in the same pattern of DNA fragments. The yield of radiation-induced apoptotic fragmentation was higher with the AVTD protocol of lysis. The novel PFGE protocol is simple and relatively non-expensive, requires only 7 h running time and gives a possibility to analyse simultaneously up to 69 samples in the same gel. The sensitivity of our protocol provides reproducible detection of 50 kb fragmentation after irradiation of human lymphocytes with 5 cGy of gamma-rays. In 2 of 6 donors tested, this DNA fragmentation was detected at dose on 2 cGy. The novel protocol can be used for quantification of 50 kb apoptotic fragments induced by different agents including low dose ionising radiations, chemicals and electromagnetic fields.     

Conformational changes in proteins in vitro as a means of predicting the acute toxicities of chemicals

Experimental data are presented on ovalbumin denaturation (OD, EC10) and human acetylcholine esterase (AChE) inhibition (IC50) in vitro, following exposure to the chemicals used in the international Multicentre Evaluation of In vitro Cytotoxicity (MEIC) programme. Data were obtained for 40 (OD test) and 43 (AChE test) of the 50 MEIC chemicals. These data were compared with similar data from other methods used in the MEIC programme, and good correlations (R2) were obtained with data from MEIC studies on cell lines: 0.80 for human, 0.81 for other animal, and 0.78 for fish cell line IC50 values and AChE values, and 0.76 for human, 0.69 other animal and 0.75 for fish cell line IC50 values and OD values. The correlation increased substantially, if chemicals which freely cross the blood-brain barrier were solely considered, with R2 = 0.90 for human, 0.90 for other animal, and 0.82 for fish cell line IC50 values and AchE values, and 0.87 for human, 0.86 for other animal, and 0.92 for fish cell line IC50 values and OD values, in this case. Such chemicals are the main cause of non-specific depression of the central nervous system (CNS). The AChE IC50 permits a good prediction of human acute toxicity, similar to the IC50 values obtained with human cell lines and the same MEIC chemicals. These results confirm the basal toxicity hypothesis formulated by Bj?rn Ekwall. It is concluded that in vitro methods based on the disruption of the functions of the proteins vital for body operation can be used as an alternative to the cell culture methods, when non-specific toxic effects of chemicals on humans and animals are evaluated.     

Involvement of p53 in gemcitabine mediated cytotoxicity and radiosensitivity in breast cancer cell lines

Gemcitabine (2',2'-difluoro-2'-deoxycytidine; dFdCyd) is one of the anti-metabolites drugs that target DNA replication. We evaluated dFdCyd cytotoxicity and its radiosensitizing ability in human breast cancer cell lines, MCF-7 (wild-type p53) and MDA-MB-231 (mutant-type p53) along with normal mammary epithelial cell line (MCF-12) for comparison. Radiosensitivity and cytotoxicity were measured by the clonogenic survival assays. DNA DSBs was studied by Pulse Field Gel Electrophoresis (PFGE) and cell cycle distribution was analyzed by flow cytometry. MDA-MB-231 cells were the most sensitive to the cytotoxicity of dFdCyd (IC(50) 5 nM) then MCF-7 (IC(50) 10nM), whereas MCF-12 cells were the most resistant to the cytotoxicity of dFdCyd (IC(50) 70 nM). MCF-12 and MCF-7 cell lines did not show any radiosensitization to dFdCyd, whereas the MDA-MB-231 cells showed significantly increased radioresistant to dFdCyd at equimolar concentration (p=0.002) and at IC(50) concentration (p<0.001). The DNA double strand breaks (DSBs) repair showed that dFdCyd neither increases DNA DSBs nor decreases the rate of their repair in MCF-12 and MCF-7 cell lines, while the same treatment in MDA-MB-231 cell line led to decrease the rate of DSBs or increase the rate of DNA repair (p=0.034). Therefore, dFdCyd is a cytotoxic agent, especially in the cancer cells irrespective of having wild-type or mutated p53 protein, but it is not effective as radiosensitizer in the cell lines used in this study. dFdCyd combined with radiation reduces the efficacy of chemo-radiotherapy in p53 mutated cells. Therefore, p53-mutated cancer could be a counter-indication for radiation-gemcitabine combined treatment.     

Microplate screening for apoptosis with antibody to single-stranded DNA distinguishes anticancer drugs from toxic chemicals

The effect of anticancer drugs and toxic compounds on cultures of human leukemic cells was evaluated by an enzyme-linked immunosorbent assay (Apoptosis ELISA) that uses a monoclonal antibody against single-stranded DNA to quantitate the apoptotic cells. The concentrations of 13 anticancer drugs, which increased Apoptosis ELISA absorbance, were close to the cytotoxic concentrations determined by the long-term cell survival assay. Short-term tetrazolium-based microculture tetrazolium (MTT) assay was significantly less sensitive than the Apoptosis ELISA and the cell survival assay for all anticancer drugs. For 6 drugs, cytotoxic concentrations measured by the MTT assay were at least 1 log higher than the concentrations inducing apoptosis. Importantly, in contrast to the anticancer drugs, 14 toxic chemicals did not increase the Apoptosis ELISA absorbance at cytotoxic concentrations. The difference in apoptosis induction by the anticancer drugs and the toxic chemicals was especially large in cultures treated with drug concentrations 2-fold higher than the IC(50) dose. Although all of the anticancer drugs tested induced intense ELISA reaction (mean absorbance 2.0), all toxic chemicals tested did not induce apoptosis. The Apoptosis ELISA assay could have useful applications in drug development as it can distinguish between clinically useful anticancer drugs and toxic compounds, has sensitivity similar to that of the long-term cell survival assay, and provides insight into the mechanism of drug cytotoxicity by differentiating between compounds killing cells by apoptosis and necrosis.     

Suppressive effects of flavonoids on dioxin toxicity

Dioxin type chemicals such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cause a variety of toxicity. Most of the toxicity of TCDD has been attributed to a mechanism by which TCDD is bound to aryl hydrocarbon receptor (AhR) and transforms the receptor. Thus, suppression of the AhR transformation by food factors can suppress the dioxin toxicity. In this study, flavonoids at various concentrations were treated to a rat cytosolic fraction containing AhR before adding 1 nM TCDD. The transformed AhR was detected by an electrophoretic mobility shift assay with a DNA oligonucleotide consensus to dioxin response element. As the results, flavones and flavonols at dietary levels act as the antagonists for AhR and suppress the transformation. The antagonistic IC50 values were in a range between 0.14 and 10 microM, which are close to the physiological levels in human. These results suggest that a plant-based diet can prevent the dioxin toxicity.     

Protein precipitation In Vitro as a measure of chemical-induced cytotoxicity: an EDIT sub-programme

As a priority area of the Evaluation-Guided Development of In Vitro Toxicity and Toxicokinetic Tests (EDIT) programme, an in vitro protein precipitation (PP) assay was used on the 50 reference chemicals of the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) project, to confirm and extend the MEIC results. Dose-response curves were generated for only 30 of the chemicals, and the concentrations causing 10% (EC10) and 50% (EC50) protein precipitation versus the positive control were chosen as endpoints. The number of chemicals with a positive response increased to 46 when a new endpoint, the minimum effect concentration (MEC) that induces protein precipitation with respect to the negative control, was used. When the results were correlated with in vitro cytotoxicity in human cell lines, a similarly good correlation was found between the various endpoints of the PP assay at 5 hours and the 24-hour IC50 average cytotoxicity in human cell lines, even though the number of chemicals included in the correlation was larger for the MEC. Using the prediction error, the endpoint that gave the best correlation between the PP assay and human cell cytotoxicity was once more found to be the 5-hour MEC, and this was chosen for the PP assay. The sensitivity of the PP assay is lower than that of the in vitro cell-line cytotoxicity assay, possibly due to its shorter exposure period and because precipitation is the ultimate event in the sequence of a protein disturbance. It is expected that earlier denaturation steps would give better sensitivity. However, this simple, inexpensive and rapid assay could be useful in the early stages of testing chemicals.     

In vitro cytotoxicity testing for prediction of acute human toxicity

This study was designed to compare the cytotoxic concentrations of chemicals, determined with three independentin vitro cytotoxicity testing protocols, with each other and with established animal LD₅₀ values, and against human toxic concentrations for the same chemicals. Ultimately, these comparisons allow us to evaluate the potential ofin vitro cell culture methods for the ability to screen a variety of chemicals for prediction of human toxicity. Each laboratory independently tested 50 chemicals with known human lethal plasma concentrations and LD₅₀ values. Two of the methods used monolayer cell cultures to measure the incorporation of radiolabeled amino acids into newly synthesized proteins and cellular protein content, while the third technique used the pollen tube growth test. The latter is based on the photometric quantification of pollen tube mass production in suspension culture. Experiments were performed in the absence or presence of increasing doses of the test chemical, during an 18- to 24-h incubation. Inhibitory concentrations were extrapolated from concentration-effect curves after linear regression analysis. Comparison of the cytotoxic concentrations confirms previous independent findings that the experimental IC₅₀ values are more accurate predictors of human toxicity than equivalent toxic blood concentrations (HETC values) derived from rodent LD₅₀s. In addition, there were no conclusive statistical differences among the methods. It is anticipated that, together, these procedures can be used as a battery of tests to supplement or replace currently used animal protocols for human risk assessment.Abbreviations DCP dichlorophenoxyacetic acid - DMEM Dulbecco's modified Eagles' medium - DMSO dimethylsulfoxide - IC inhibitory concentration - LD₅₀ lethal dose 50% - MEIC Multicenter Evaluation forIn Vitro Cytotoxicity - PI₅₀ protein inhibition 50% - PTG pollen tube growth - TCA trichloroacetic acid - TCE trichloroethane     

Increased frequency of sister-chromatid exchange and chromatid breaks in lymphocytes after treatment of human volunteers with therapeutic doses of paracetamol

Paracetamol was given to 10 healthy human volunteers in 3 doses of 1 g each during a period of 8 h. Blood samples for lymphocyte cultures were taken before and 24 h after paracetamol administration. A small but significant increase was found in the frequency of sister-chromatid exchanges (SCE) after intake of paracetamol (0.187 +/- 0.030 per chromosome before and 0.208 +/- 0.024 per chromosome after). After exposure the mean frequency of chromatid breaks per 100 cells was significantly increased (2.16 +/- 1.33 versus 0.33 +/- 0.50 before exposure). Exposure of human lymphocytes in vitro showed that concentrations of paracetamol above 0.1 mM induced inhibition of replicative DNA synthesis. Increased SCE was found in lymphocytes exposed to 1-10 mM paracetamol for 2 h. Furthermore, 0.75-1.5 mM paracetamol exposure for 24 h increased the frequency of chromatid and chromosome breaks in the lymphocytes. The paracetamol-induced SCE and chromosome aberrations may be secondary effects of paracetamol-induced inhibition of DNA synthesis or due to covalent binding of paracetamol metabolite(s) to DNA.     

Lead and catechol hematotoxicity in vitro using human and murine hematopoietic progenitor cells

In vitro cloning assays for hematopoietic myeloid and erythroid precursor cells have been used as screening systems to investigate the hematotoxic potential of environmental chemicals in humans and mice. Granulocyte-monocyte progenitors (CFU-GM) from human umbilical cord blood and from mouse bone marrow (Balb/c and B6C3F1) were cultured in the presence of lead and the benzene metabolite catechol. Erythroid precursors (BFU-E) from human umbilical cord blood were cultured in the presence of lead. The in vitro exposure of the human and murine cells resulted in a dose-dependent depression of the colony numbers. The concentration–effect relationship was studied. Results showed that: (1) Based on calculated IC50 values, human progenitors are more sensitive to lead and catechol than are murine progenitors. The dose that caused a 50% decrease in colony formation after catechol exposure was 6 times higher for murine cells (IC50 = 24 μmol/L) than for human cord blood cells (IC50 = 4 μmol/L). Lead was 10–15 times more toxic to human hematopoietic cells (IC50 = 61 μmol/L) than to murine bone marrow cells from both mice strains tested (Balb/c, IC50 = 1060 μmol/L; B6C3F1, IC50 = 536 μmol/L). (2) A lineage specificity was observed after exposure to lead. Human erythroid progenitors (hBFU-E) (IC50 = 3.31 μmol/L) were found to be 20 times more sensitive to the inhibitory effect of lead than were myeloid precursors (hCFU-GM) (IC50 = 63.58 μmol/L). (3) Individual differences in the susceptibility to the harmful effect of lead were seen among cord blood samples. (4) Toxicity of lead to progenitor cells occurred at environmentally relevant concentrations. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genotoxic effects of paracetamol in V79 Chinese hamster cells

Paracetamol was studied for possible genotoxic effects in V79 Chinese hamster cells. Paracetamol (0.5 mM for 30 min) reduced the rate of DNA synthesis in exponentially growing V79 cells to about 50% of control. A further decrease in the DNA synthesis was seen during the first 30 min after termination of paracetamol exposure. Paracetamol (3 and 10 mM for 2 h) caused a small increase in DNA single-strand breaks, as measured by the alkaline elution technique. After 16 h elution, the amount of DNA retained on the filters was 79 and 70% of controls in cells treated with 3 and 10 mM paracetamol respectively. No indication of DNA damage was seen in measuring the effect of paracetamol (0.25-10 mM for 2 h) on unscheduled DNA synthesis in growth-arrested cultures of V79 cells. At the highest concentrations (3 and 10 mM paracetamol), decreased unscheduled DNA synthesis was observed. Also UV-induced DNA-repair synthesis was inhibited by 3 and 10 mM paracetamol. DNA-repair synthesis was, however, inhibited at a much higher concentration than that inhibiting replicative DNA synthesis. The number of sister-chromatid exchanges (SCE) increased in a dose-dependent manner on 2 h exposure to paracetamol from 1 mM to 10 mM. At the highest dose tested (10 mM), the number of SCE increased to 3 times the control value. Co-culturing the V79 cells with freshly isolated mouse hepatocytes had no further effect on the paracetamol induced sister-chromatid exchanges. The present study indicates that paracetamol may cause DNA damage in V79 cells without any external metabolic activation system added.     

Relative efficiencies of the bacterial, yeast, and human DNA methyltransferases for the repair of O6-methylguanine and O4-methylthymine. Suggestive evidence for O4-methylthymine repair by eukaryotic methyltransferases

The suicidal inactivation mechanism of DNA repair methyltransferases (MTases) was exploited to measure the relative efficiencies with which the Escherichia coli, human, and Saccharomyces cerevisiae DNA MTases repair O6-methylguanine (O6MeG) and O4-methylthymine (O4MeT), two of the DNA lesions produced by mutagenic and carcinogenic alkylating agents. Using chemically synthesized double-stranded 25-base pair oligodeoxynucleotides containing a single O6MeG or a single O4MeT, the concentration of O6MeG or O4MeT substrate that produced 50% inactivation (IC50) was determined for each of four MTases. The E. coli ogt gene product had a relatively high affinity for the O6MeG substrate (IC50 8.1 nM) but had an even higher affinity for the O4MeT substrate (IC50 3 nM). By contrast, the E. coli Ada MTase displayed a striking preference for O6MeG (IC50 1.25 nM) as compared to O4MeT (IC50 27.5 nM). Both the human and the yeast DNA MTases were efficiently inactivated upon incubation with the O6MeG-containing oligomer (IC50 values of 1.5 and 1.3 nM, respectively). Surprisingly, the human and yeast MTases were also inactivated by the O4MeT-containing oligomer albeit at IC50 values of 29.5 and 44 nM, respectively. This result suggests that O4MeT lesions can be recognized in this substrate by eukaryotic DNA MTases but the exact biochemical mechanism of methyltransferase inactivation remains to be determined.     

A non-radioactive, PFGE-based assay for low levels of DNA double-strand breaks in mammalian cells

A PFGE method was adapted to measure DNA double-strand breaks (DSBs) in mammalian cells after low (0-25 Gy) doses of ionising radiation. Instead of radionuclide incorporation, DNA staining in the gel by SYBR-Gold was used, which lowered the background of DNA damage and could be applied to non-cycling cells. DSB level was defined as a product of a fraction of DNA released to the gel (FR) and a number of DNA fragments in the gel (DNA(fragm)) and expressed as a percentage above control value. The slope of the dose-response curve was two-fold higher compared to that with FR alone as DSB level indicator (31.4 versus 15.6% per Gy). Two alternative ways were proposed to determine the total amount of DNA, used for FR calculation: measurement of DNA content in a plug not subjected to electrophoresis, with the use of Pico-Green, or estimation of DNA released to the gel from a plug irradiated with 600 Gy of gamma-rays. The limit of DSB detection was 0.25 Gy for human G1-lymphocytes and 0.5-1 Gy for asynchronous cultures of human glioma M059 K and J or mouse lymphoma L5178Y-R and -S cells. Specificity of our PFGE assay to DSB was confirmed by the fact that no damage was detected after treatment of the cells with H(2)O(2), an inducer of single-strand DNA breaks (SSBs). On the contrary, the H(2)O(2) inflicted damage was detected by neutral comet assay, attaining 160% above control (equivalent to 2.5 Gy of X-radiation). DSB rejoining, measured in cells after X-irradiation with a dose of 10 Gy, generally proceeded faster than that measured previously after higher (30-50 Gy) doses of ionising radiation. Clearly seen were defects in DSB rejoining in radiosensitive M059 J and L5178Y-S cells compared to their radioresistant counterparts, M059 K and L5178Y-R. In some cell lines, a secondary post-irradiation increase in DSB levels was observed. The possibility is considered that these additional DSBs may accumulate during processing of non-DSB clustered DNA damage or/and represent early apoptotic events.     

Design and synthesis of benzo[c,d]indolone-pyrrolobenzodiazepine conjugates as potential anticancer agents

A series of benzo[c,d]indol-2(1H)one-PBD conjugates (11a-l) have been designed and synthesized as potential anticancer agents. These compounds were prepared by linking the C8-position of DC-81 with a benzo[c,d]indol-2(1H)one moiety through different alkane spacers in good yields and confirmed by (1)H NMR, mass and HRMS data. The DNA binding ability of these conjugates was evaluated by thermal denaturation studies and interestingly, compound 11l showed enhanced DNA binding ability. These compounds were also evaluated for their anticancer activity in selected human cancer cell lines of lung, skin, colon and prostate by using MTT assay method. These new conjugates showed promising anticancer activity with IC(50) values ranging from 1.05 to 36.49 μM. Moreover, cell cycle arrest in SubG1 phase was observed upon treatment of A549 cells with 1 and 2 μM (IC(50)) concentrations of compound 11l and it induced apoptosis. This is confirmed by Annexin V-FITC, Hoechst staining, caspase-3 activity as well as DNA fragmentation analysis.     

Elucidation of strict structural requirements of brefeldin A as an inducer of differentiation and apoptosis

Brefeldin A (BFA) can induce a wide variety of human cancer cells to differentiation and apoptosis and is in development as an anticancer agent. To elucidate structural requirements for cytotoxicity and induction of differentiation and apoptosis, BFA was structurally modified into various derivatives including 4-epi-BFA in this study. Their inducing activities of apoptosis were evaluated with their cytotoxicities, DNA fragmentation and morphological changes in human colon cancer cell HCT 116. The cytotoxicity of 4-epi-BFA (TX-1923) (IC50 = 60 microM) was 300 times lower than that of BFA (IC50 = 0.2 microM). Furthermore, 4-epi-BFA induced DNA fragmentation and apoptotic morphological changes at much higher concentrations (70 and 50 microM, respectively) than BFA (0.11 and 0.36 microM, respectively). These results indicated that the configuration of 4-hydroxyl group of brefeldin A plays a key role in the cytotoxicity and induction of apoptosis. On the other hand, 7-O-acetyl-BFA, 4-O-acetyl-BFA, and 4,7-di-O-acetyl-BFA exhibited potential activities in cytotoxicity and inducibility of apoptosis. We suggested that the structural determinants for BFA include the moiety of the Michael acceptor, the conformational rigidity of the 13-membered ring, and the configuration of 4-hydroxyl group.     

DNA oxidatively damaged by chromium(III) and H(2)O(2) is protected by the antioxidants melatonin, N(1)-acetyl-N(2)-formyl-5-methoxykynuramine, resveratrol and uric acid

Chromium (Cr) compounds are widely used industrial chemicals and well known carcinogens. Cr(III) was earlier found to induce oxidative damage as documented by examining the levels of 8-hydroxydeoxyguanosine (8-OH-dG), an index for DNA damage, in isolated calf thymus DNA incubated with CrCl(3) and H(2)O(2). In the present in vitro study, we compared the ability of the free radical scavengers melatonin, N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK), resveratrol and uric acid to reduce DNA damage induced by Cr(III). Each of these scavengers markedly reduced the DNA damage in a concentration-dependent manner. The concentrations that reduced 8-OH-dG formation by 50% (IC(50)) were 0.10 microM for both resveratrol and melatonin, and 0.27 microM for AFMK. However, the efficacy of the fourth endogenous antioxidant, i.e. uric acid, in terms of its inhibition of DNA damage in the same in vitro system was about 60--150 times less effective than the other scavengers; the IC(50) for uric acid was 15.24 microM. These findings suggest that three of the four antioxidants tested in these studies may have utility in protecting against the environmental pollutant Cr and that the protective effects of these free radical scavengers against Cr(III)-induced carcinogenesis may relate to their direct hydroxyl radical scavenging ability. In the present study, the formation of 8-OH-dG was likely due to a Cr(III)-mediated Fenton-type reaction that generates hydroxyl radicals, which in turn damage DNA. Once formed, 8-OH-dG can mutate eventually leading to cancer; thus the implication is that these antioxidants may reduce the incidence of Cr-related cancers.     

Synergistic action of tiazofurin and difluorodeoxycytidine on differentiation and cytotoxicity.

Tiazofurin (TR), an inhibitor of IMP dehydrogenase, causes remissions and induced differentiation in human leukemia through lowering the concentrations of GTP and dGTP. A deoxycytidine analog, difluorodeoxycytidine (DFDC), is an anti-tumor agent phosphorylated by deoxycytidine kinase, resulting in decreased concentration of dCTP, leading to inhibition of DNA synthesis. In HL-60 cells DFDC induced differentiation and inhibited proliferation in a dose-dependent manner (IC50 = 4 nM); TR provided synergism with DFDC. DFDC inhibited proliferation in OVCAR-5 human ovarian carcinoma cells (IC50 = 25 nM) and colony formation in PANC-1 human pancreatic carcinoma cells (IC50 = 2 nM) and rat hepatoma 3924A cells (IC50 = 22 nM). TR and DFDC are synergistically cytotoxic in hepatoma cells and additive in PANC-1 cells. The two drugs together should be helpful in treating leukemias and solid tumors in humans.     

Evaluation of HI-6 oxime: potential use in protection of human acetylcholinesterase inhibited by antineoplastic drug irinotecan and its cyto/genotoxicity in vitro

The function of acetylcholinesterase (AChE) is the rapid hydrolysis of the neurotransmitter acetylcholine (ACh), which is involved in the numerous cholinergic pathways in both the central and the peripheral nervous system. Therefore, AChE measurement is of high value for therapy management, especially during the course of intoxication with different chemicals or drugs that inhibit the enzyme. Pyridinium or bispyridinium aldoximes (oximes) are able to recover the activity of the inhibited enzyme. Since their adverse effects are not well elucidated, in this study the efficiency of HI-6 oxime in protection and/or reactivation of human erythrocyte AChE inhibited by the antineoplastic drug irinotecan as well as its cyto/genotoxicity in vitro were investigated. HI-6 was effective in protection of AChE and increased its activity up to 30%; the residual activity after irinotecan inhibition was 7%. Also, it reactivated the enzyme previously inhibited by 50% irinotecan (4.6 microg/ml) applied at 1/4 of the IC50 value. The tested concentrations of HI-6 exhibited acceptable genotoxicity towards white blood cells, as estimated by the alkaline comet assay, DNA diffusion assay and cytogenetic endpoints (structural chromosome aberrations and cytokinesis-block micronucleus assay). The results obtained warrant the further investigation of HI-6 in vivo, as well as its development for possible application in chemotherapy.     

Cytotoxicity of the MEIC reference chemicals in antioxidant-enriched, rat hepatoma-derived Fa32 cells

Since vitamin E increases the antioxidant status of cells, its influence on cytotoxicity was investigated. The neutral red uptake (NRU) inhibition effects of 39 MEIC reference chemicals were measured after treatment of rat hepatoma-derived Fa32 cells in the presence of vitamin E for 30 minutes. The results were quantified in terms of the NI50, the concentration of test compound required to reduce the NRU by 50%. Sodium chloride was the only chemical that was more toxic in the presence of vitamin E. This effect was related to the concentration of vitamin E in the cell culture medium. A vitamin E dose-related response was also observed for the decreased toxicity of paracetamol and caffeine. Glutathione levels were slightly increased in the presence of vitamin E, which could contribute to the protective effect of vitamin E. Of the remaining chemicals, 50% were less toxic in the presence of vitamin E, but the correlation with the acute human toxicity data of the MEIC study was not improved. The results imply that reactive oxygen species interfere with the toxicity of a high proportion of toxic chemicals. The assay described provides a quick and easy method for checking whether reactive oxygen species contribute to the toxicity of a chemical.