Single Cell Microgel Electrophoresis Assay Research Articles

Chlorophyll derivative mediated PDT versus methotrexate: An in vitro study using MCF-7 cells

Breast cancer is the most common cause of cancer deaths among women worldwide. Although chemotherapy is a standard method for the treatment of breast cancer, the photodynamic therapy (PDT) is a recent promising modality for cancer diagnosis and treatment. Its major advantages over chemotherapy are better selectivity of tumour tissue destruction and lack of severe local and systemic complications. This work is directed towards evaluation of the efficacy of Photodynamic therapy using chlorophyll derivative (CHL) as a photosensitizer in treatment of breast cancer. It also aims at investigation of the genetic safety of chlorophyll mediated PDT in comparison to the conventional chemotherapy. Both methotrexate (MTX) and light activated chlorophyll derivative were used to target MCF-7 breast cancer cell line. Standard karyotyping and alkaline single cell microgel electrophoresis assay (Comet assay) were applied on normal human peripheral blood lymphocytes (HPL) in order to investigate the respective possible mutagenic and genotoxic side effects that might result from application of each therapeutic modality. Results obtained from this study showed that 50% of MCF-7 tumour cell death (LC(50)) was reached by using a concentration of chlorophyll derivative that is 138 times lower than MTX. Moreover, chlorophyll derivative exerted no genetic side effects as compared to MTX that resulted into several types of chromosomal breakages. Compared to MTX, light activated chlorophyll derivative proved to be a better candidate for breast cancer cell toxicity, referring to its higher efficacy at tumour cells killing, safety to normal cells and simple method of extraction.

Cumulative genotoxic and apoptotic effects of xenobiotics in a mini organ culture model of human nasal mucosa as detected by the alkaline single cell microgel electrophoresis assay and the annexin V-affinity assay

Three-dimensional mini organ cultures of human inferior nasal turbinate epithelia have proved to be a useful tool in genotoxicology studies. They allow repetitive or chronic exposure of cells to xenobiotics in a well-preserved organ-specific mucosal architecture for an extended period of time. It is the aim of the present study to concurrently monitor cumulative genotoxic and apoptotic effects of sodium dichromate, N-nitrosodiethylamine (NDEA) and N-methyl- N-nitro- N-nitroso-guanidine (MNNG). Mini organs were raised by separating fresh specimens of human inferior nasal turbinates ( n = 11) into 1 mm 3 sized pieces and culturing them on multiwell plates with bronchial epithelial basal medium for 6 days. Aliquots of the mini organs were subsequently exposed to sodium dichromate (1.0 mM, 1 h), NDEA (50 mM, 1 h) or MNNG (0.07 mM, 1 h) on days 7, 9 and 11 versus a single exposure on day 11 only. DNA fragmentation and apoptotic events were assessed on day 11 using the alkaline single cell microgel electrophoresis assay (comet assay) and the annexin V-affinity assay. Significant DNA fragmentation could be demonstrated after a single exposure of the mini organs to sodium dichromate. Following three subsequent incubations, there was a further increase in the genetic damage observed, accompanied by an increase in the rate of apoptotic cells. In contrast, after single and triple incubation with NDEA there was neither an increase in genetic damage nor in the fraction of apoptotic cells detectable. Repetitive exposure to MNNG resulted in an accumulation of DNA damage without an observable increase in apoptosis. The results verify the need to assess apoptosis in genotoxicology research and to investigate cumulative effects of xenobiotics. Three-dimensional mini organ cultures of human upper aerodigestive tract epithelia have shown to be well-suited for improving the ability to distinguish between cumulative genotoxic and apoptotic effects.

Cloudy apple juice decreases DNA damage, hyperproliferation and aberrant crypt foci development in the distal colon of DMH-initiated rats

Clear (CleA) and cloudy (CloA) apple juices containing different amounts of analyzed procyanidins and pectin were investigated for preventive effects of colon cancer and underlying molecular mechanisms in F344 rats given intraperitoneal injections of 1,2-dimethylhydrazine (DMH; 20 mg/kg body wt) once a week for 4 weeks. Rats received either water (Cont), CleA or CloA (ad libitum) for 7 weeks starting 1 week before the first DMH injection. CloA inhibited DMH induced genotoxic damage in mucosa cells of the distal colon compared with Cont as investigated by single-cell microgel electrophoresis assay. The mean tail intensity in mucosa cells of DMH-treated controls (Cont/DMH: 6.1+/-0.9%) was significantly reduced by CloA (2.4+/-0.8%; P<0.01) but not by CleA intervention (4.1+/-1.2%; P>0.05). The crypt cell proliferation index induced by DMH (Cont/NaCl: 10.0+/-0.7%; Cont/DMH: 19.9+/-1.0%; P<0.001) was significantly decreased by CleA (15.7+/-0.7%; P<0.001) and CloA intervention (11.9+/-0.4%; P<0.001). CloA but not CleA significantly reduced the number of large aberrant crypt foci (ACF) consisting of more than four aberrant crypts (AC) (Cont/DMH: 37.4+/-5.4; CleA/DMH: 32.8+/-4.4, P>0.05; CloA/DMH: 18.8+/-2.5 ACF; P<0.05) and the overall mean ACF size in the distal colon (Cont/DMH: 2.31+/-0.09; CleA/DMH: 2.27+/-0.05; CloA/DMH: 2.04+/-0.03 AC/ACF; P<0.05). After treatment with DMH and/or apple juices there were no changes in transcript levels of colonic cyclooxygenase isoforms (COX-1, COX-2) or glutathione-associated enzymes (GST-M2, gamma-GCS, GST-P), the splenocyte natural killer cell activity and plasma antioxidant status. However, CloA but not CleA prevented the DMH-induced reduction of splenocyte CD4/CD8 (T-helper cells to cytotoxic lymphocytes) ratio. Since both formulations contained comparable concentrations and types of monomeric polyphenols, complex polyphenols or non-polyphenolic compounds, such as pectin might be responsible for the stronger cancer-preventive effect by CloA.

Lack of an association between environmental exposure to polychlorinated biphenyls and p,p'-DDE and DNA damage in human sperm measured using the neutral comet assay.

Chlorinated organic chemicals, such as polychlorinated biphenyls (PCB), hexachlorobenzene (HCB), dichlorodiphenyl trichloroethane (DDT), and dichlorodiphenyl dichloroethene (DDE, the most stable daughter compound of DDT) are persistent lipophilic compounds found in a large portion of the general population. To explore the hypothesis that environmental exposure to these compounds is associated with altered DNA integrity in human sperm, a study of 212 male partners of a sub-fertile couple who presented to the Massachusetts General Hospital Andrology Laboratory was conducted. The neutral single cell microgel electrophoresis assay (comet assay) was used to assess DNA integrity in sperm. VisComet image analysis software was used to measure total comet length, the proportion of DNA present in the comet tail, and tail distributed moment, an integrated measure of length and intensity. In the regression analyses, there were no statistically significant consistent associations between the comet assay parameters and any of the individual PCB congeners, sum of PCB, or p,p'-DDE. These results suggest that there are not strong relationships between adult levels of these chlorinated organic compounds and sperm DNA damage as measured by the comet assay.

The relationship between environmental exposures to phthalates and DNA damage in human sperm using the neutral comet assay.

Phthalates are industrial chemicals widely used in many commercial applications. The general population is exposed to phthalates through consumer products as well as through diet and medical treatments. To determine whether environmental levels of phthalates are associated with altered DNA integrity in human sperm, we selected a population without identified sources of exposure to phthalates. One hundred sixty-eight subjects recruited from the Massachusetts General Hospital Andrology Laboratory provided a semen and a urine sample. Eight phthalate metabolites were measured in urine by using high-performance liquid chromatography and tandem mass spectrometry; data were corrected for urine dilution by adjusting for specific gravity. The neutral single-cell microgel electrophoresis assay (comet assay) was used to measure DNA integrity in sperm. VisComet image analysis software was used to measure comet extent, a measure of total comet length (micrometers); percent DNA in tail (tail%), a measure of the proportion of total DNA present in the comet tail; and tail distributed moment (TDM), an integrated measure of length and intensity (micrometers). For an interquartile range increase in specific gravity-adjusted monoethyl phthalate (MEP) level, the comet extent increased significantly by 3.6 micro m [95% confidence interval (95% CI), 0.74-6.47]; the TDM also increased 1.2 micro m (95% CI, -0.05 to 2.38) but was of borderline significance. Monobutyl, monobenzyl, monomethyl, and mono-2-ethylhexyl phthalates were not significantly associated with comet assay parameters. In conclusion, this study represents the first human data to demonstrate that urinary MEP, at environmental levels, is associated with increased DNA damage in sperm.

N-nitrososdimethylamine is activated in microsomes from hepatocytes to reactive metabolites which damage DNA of non-parenchymal cells in rat liver

The liver carcinogen N-nitrosodimethylamine (NDMA) has to be metabolically activated by specific cytochromes before it can react with cellular macromolecules (e.g. proteins or DNA). Although hepatocytes are believed to be responsible for this activation, the liver tumours originate mainly from non-parenchymal cells (NPC). To investigate their activation capacity we determined NDMA-demethylase activity in isolated microsomes from both liver cell types. The results demonstrate that only hepatocytes have activation capacity. Additional experiments were performed with hepatocytes and NPC using the single cell microgel electrophoresis assay (MGE). DNA damage appears in both cell types following in vivo exposure. Tested in vitro, however, the carcinogens induce DNA damages only in hepatocytes (the cells which activate these compounds). N-nitroso-hydroxymethyl-methylamine could be the responsible metabolite as it is stable enough to be transported from hepatocytes to NPC in an intact liver.

Cytotoxicity and genotoxicity of fluorides in human mucosa and lymphocytes

Fluorides are widely used in dental health products and drinking water, due to their beneficial effects in caries-prophylaxis and -treatment. Nevertheless, irritation of the gingiva and oropharyngeal mucosa as well as in gastric mucosa is observed since neither local nor systemic application is restricted to the teeth. These effects may partly be attributed to a known cytotoxicity of fluorides. Whether fluorides also have genotoxic effects on human mucosa or lymphocytes as a possible factor in tumor initiation was investigated in this study. Human oropharyngeal epithelial cells and peripheral lymphocytes were incubated after single cell preparation with the aminefluoride Olaflur at concentrations of 2 ppm, 21 ppm, 35 ppm, 71 ppm and 213 ppm. The extent of cytotoxicity was investigated using the trypan blue exclusion test. Following incubation, electrophoresis for migration of DNA fragments, fluorescence staining and digital image analysis according to a standard protocol of the single cell microgel electrophoresis assay (Comet assay) followed. DNA damage was characterized using the Olive Tail Moment (OTM). For fluoride concentrations of 2 ppm to 35 ppm, non vital cells of less than 10% could be shown. After incubation with 71 ppm and 213 ppm Olaflur, there were 15% and 43% of damaged cells, respectively. Weak genotoxic effects on mucosal cells as well as on lymphocytes could be demonstrated at all concentrations tested. In fluoride concentrations of 213 ppm genotoxicity increased to max. OTM-levels of 23. Beside the cytotoxic effect of fluorides, also a minor genotoxic impact on human mucosa and on peripheral lymphocytes could be demonstrated using the Comet assay. Further investigations are warranted to examine fluorides in a model allowing for repeated or long term incubations on structurally intact human mucosa in vitro. Such a model will help to distinguish between DNA damage that may be repaired successfully and other impairments that may show an additive character in repetitive or chronic exposure in vivo.

Mutagen sensitivity of nasopharyngeal cancer patients

Primary nasopharyngeal carcinomas (NPCs) may be of various types, including squamous cell carcinomas, undifferentiated carcinomas, and lymphoepitheliomas. Tumor initiation has been linked to the Epstein-Barr virus and, in some geographical regions, to alimentary factors. Possible hereditary components for the appearance of NPCs have not yet been clearly identified. In this study, genetic sensitivity to the genotoxic effects of carcinogenic xenobiotics as an endogenous risk factor of tumor initiation was investigated. The single cell microgel electrophoresis assay was used to quantify chemically-induced DNA damage in lymphocytes of 30 NPC patients and 30 non-tumor donors. The xenobiotics investigated were N′-nitrosodiethylamine, sodium dichromate, and nickel sulphate, with N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) and dimethyl sulfoxide (DMSO) as positive and negative controls, respectively. The extent of DNA migration in the solvent control cultures was not significantly different between the two groups (1.2±0.5 mean Olive tail moment and standard deviation of 30 individuals for NPC patients; 1.1±0.4 for non-tumor donors). With constant exposure and electrophoretic conditions, genotoxic effects of varying degrees were induced by the different xenobiotics in tumor and non-tumor patients (nickel sulphate: 7.1±2.5 for NPC patients and 5.9±1.6 for non-tumor donors; sodium dichromate: 18.1±5.3 for NPC patients and 16.2±5.4 for non-tumor donors; MNNG: 47.8±13.3 for NPC patients and 52.7±13.6 for non-tumor donors). Only N′-nitrosodiethylamine proved to induce significantly more DNA migration in lymphocytes of tumor patients (9.8±3.1) as compared to non-tumor patients (8.2±2.3). Although for sodium dichromate the degree of DNA migration did not significantly differ, variability in migration patterns proved to be lower in the tumor group. Mutagen sensitivity of NPC patients was shown to be elevated for a selected xenobiotic, whereas a general elevation of DNA fragility was not present. Further studies on mutagen sensitivity as an endogenous risk factor influencing the susceptibility of patients at the time of first diagnosis of nasopharyngeal carcinomas are warranted.

Inter- and intra-individual variation of faecal water — genotoxicity in human colon cells

Exogenous nutritional factors modulate the faecal contents leading to an enhanced or reduced burden with toxic and cancerogenic factors. These factors are thought to contribute to colon cancer by inducing mutations or enhancing proliferation in colon cells. Faecal water more or less causes these effects in model systems and thus could be the basis for valuable biomarker approaches. Our investigations are aimed at determining geno- and cytotoxicity of faecal water in human colon cell lines in vitro. We are developing techniques for their applicability as biomarker tests during dietary intervention studies. Faecal water is isolated by centrifugation of the faeces at 25 000×g and added to cultured human colon cells (HT29). Membrane damage as assessed by trypan blue exclusion is determined as a measure for cytotoxicity. Semiquantitative analysis of inducible DNA damage (breaks and alkali labile sites) are analysed with the single cell microgelelectrophoresis assay (comet-assay) and oxidised DNA bases by the additional use of repair specific enzymes. We have now determined baseline toxic activities and calculated inter- and intra-individual and -experimental coefficients of variation for faecal water from different subjects consuming similar or different diets. Most faecal water induced DNA damage and oxidised DNA bases in HT29 clone 19a cells (0.9–9.14 fold and 1.7–4.9 fold, respectively in comparison to the NaCl controls). Intra- and inter-experimental coefficients (CV) of variation, were in a similar order of magnitude and ranged from 6.9 to 31.4. In contrast both intra- and inter-individual variability were considerably higher (CV-ranges of 29.7–76.6 and 21.3–64.0, respectively). Interestingly, these inter-individual values were not lowered when subjects consumed identical diets (CV-ranges of 28.4–126.0). However, following intervention with certain protective dietary regimens (e.g. lignan containing bread) significant reductions of faecal water-induced genotoxicity can be observed. Therefore, in spite of the expected and observed degrees of variation in this methodology, effective experimental protocols may still lead to detectable modulations of the level of toxic and genotoxic effects.

On the mechanism of cogenotoxic action between ingested amphibole asbestos fibres and benzo[a]pyrene: II. Tissue specificity studies using comet assay

Epidemiological data seem to be equivocal on the probable increase in cancer incidence in populations exposed to asbestos-fibre contaminated drinking water. Although animal experiments failed to demonstrate carcinogenicity of oral asbestos exposure, the large surface area of the fibres, however, creates the possibility of cogenotoxic action with adsorbed water-borne organics. In our animal model, rats were gavaged with untreated UICC crocidolite and anthophyllite fibres and fibres that had been allowed to adsorb benzo[a]pyrene molecules from aqueous solutions. Peritoneal macrophages and intestine, parietal peritoneum and omentum samples were obtained from the animals after 24 h. The alkaline single-cell microgel electrophoresis assay (comet assay) was performed on cells isolated from the solid tissues. Tail moment was applied as a basis of evaluation following image analysis. Our results indicate high levels of DNA strand breaks in the cells prepared from the omentum and intestine. We could also demonstrate a significant potentiating effect of the adsorbed carcinogen on the induction of DNA damage in the omentum. The parietal peritoneum and macrophages were not involved in the early genotoxic alterations under study. Our results support the molecular model of asbestos cocarcinogenesis, including both asbestos-induced deletions and mutations caused by a mutagen carried by the same fibres.

Genotoxische Wirkung auf menschliche Schleimhautbiopsien des oberen Aerodigestivtraktes

In numerous epidemiologic studies, environmental and occupational substances such as sodium dichromate (Na2Cr2O7), benzo[a]pyren (B(a)P), and N'nitroso-diethanolamine (NDELA) have been shown to be of potential carcinogenic risk on human epithelial cells in the upper aerodigestive tract. Using the alkaline microgel electrophoresis technique (comet assay). mucosal cells isolated from biopsies of the upper aerodigestive tract (nose, paranasal sinuses, mouth, pharynx, larynx, and tonsils) were used to analyze target sites for different genotoxic substances and specific sensitivities of each donor. The cells were freshly isolated by enzymic digestion. 0.5-1 x 10(6) cells per donor were obtained with viabilities between 80-100%. After in vitro incubation, the cells were subsequently subjected to the single cell microgel electrophoresis assay. Results were evaluated regarding the personal history of each donor, focusing on previous exposure to tobacco, alcohol, and occupational compounds. Na2Cr2O7 induced strong genotoxic damage in the nasal and paranasal sinus epithelia as well as in mucosa cells of the larynx. NDELA caused significant damage in mouth cavity epithelia and showed also to be harmful towards mucosa of pharynx and larynx. B(a)P induced fewer DNA strand breaks in mucosal cells of mouth, pharynx and larynx. Significant differences between individuals were apparent for tissue samples from different donors. The genotoxic damage induced in cells of donors with a history of chronic alcohol consumption was significantly higher than in cells of patients without chronic abuse of alcohol. The data shows that DNA damage in human epithelial cells of the upper aerodigestive tract induced by environmental and occupational substances can be demonstrated using the microgel electrophoresis technique. The influence of chronic alcohol consumption on the genotoxic effects of substances such as NDELA and B(a)P showed the importance of evaluating preexisting compounding factors.

Genotoxic bifunctional aldehydes produce specific images in the comet assay

Seven genotoxic aldehydes (acrolein, chloroacetaldehyde, crotonaldehyde, formaldehyde, glutardialdehyde, glyoxal, and methylacrolein) have been studied in vitro using the alkaline version of the comet assay (or single cell microgel electrophoresis assay) in freshly isolated rat hepatocytes. Chloroacetaldehyde, glyoxal and methylacrolein treatment resulted in an elevated tail moment (TM), used as indicator for an DNA damaging activity and formation of comet like structures. In addition, this treatment also caused characteristic DNA spot images with small, highly condensed areas within the otherwise circular DNA spots. These were not seen in solvent and N-Methyl- N′-nitro- N-nitrosoguanidine (MNNG)-treated control cells. Treatment of hepatocytes with acrolein, crotonaldehyde, formaldehyde and glutardialdehyde resulted in an TM which did not differ from those of control values although 86–95% of the cells showed characteristic changes of their DNA spot images. The condensed areas are probably the consequence of the known DNA and protein crosslinking activities of these bifunctional aldehydes. It is suggested that using the alkaline comet assay both TM (or overall comet length) as well as changes in the DNA spot image should be evaluated.

In vivo studies on genotoxicity of pure and commercial linuron

The ureic herbicide linuron [3-(3,4-dichlorophenyl)-1-methoxy-1-methylurea] (CAS 330-55-2) was investigated for genotoxicity in a series of in vivo experiments. Since human exposure to herbicides is not only to the active principles, but also to all the chemicals present in the commercial formulation, we tested both pure and commercial linuron. Groups of rats were treated with gavage containing different doses of the herbicide (pure compound or commercial formulation) for 14 days. The doses were 150, 300 and 450 mg/kg b.wt. for the pure compound and 315.8, 631.6 and 947.4 mg/kg b.wt. for the commercial formulation (47.5% of linuron). Faeces and urine were collected at regular intervals. Urine specimens were analysed for their mutagenic metabolites, thioethers and d-glucaric acid content. Faeces extracts were tested for mutagenicity. Linuron's ability to cause DNA damage and cytogenetic effects was also investigated after treating groups of rats once with different doses of pure or commercial linuron. DNA single-strand breaks were assessed in rat liver using the alkaline elution technique and the single-cell microgel electrophoresis assay (SCGE: `comet' assay), and in rat testes cells with the SCGE assay. Micronuclei induction was analysed in rat bone marrow erythrocytes. Results obtained were mainly negative when the excretion of mutagenic metabolites in urine and faeces of animals treated with the pure compound or with the linuron-based commercial formulation were monitored, whereas an increase in the urinary excretion of thioethers and d-glucaric acid was observed in rats treated with the commercial formulation. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the treated animals. However, linuron affected the viability of hepatocytes isolated from animals treated with higher doses. This cytotoxicity was accompanied by the induction of DNA single-strand breaks in the liver, as seen by the alkaline elution assay. The potential of pure linuron to induce in vivo DNA damage was confirmed with the microgel electrophoresis technique (`comet' assay). Cytotoxicity was also seen in rat testes cells. However, no indication of DNA damage was visible.

Evaluation of manual and image analysis quantification of DNA damage in the alkaline comet assay.

The alkaline comet assay or single cell microgel electrophoresis assay is a sensitive method of detecting DNA strand breaks and alkali labile sites in individual cells. The results of this assay can be analysed by different methods. In this study we compared analyses of the same slides by a manual method and by image analysis, post-treatment of clone 707 Friend erythroleukaemia cells with H2O2. The parameters which were found to be particularly useful were comet area and comet length (measured manually) and percentage tail DNA, tail moment, tail length and tail length/head radius (L/H), measured using image analysis. The manual method for comet analysis presented in this paper would appear to provide good and reliable comet data. However, the image analysis comet system described offers an alternative analysis method which avoids the need for photomicrographs and tedious manual analysis. The image analysis parameters: % tail DNA, tail moment, tail length and L/H give good consistent results and for large-scale analysis it will, therefore, conceivably be the method of choice.

Transport of reactive metabolites of procarcinogens between different liver cell types, as demonstrated by the single cell microgel electrophoresis assay

Procarcinogens have to be activated by specific cytochromes before showing adverse effects. Freshly isolated hepatocytes (parenchymal liver cells, PC) are characterized by a high content of such xenobiotic enzymes and are widely used to investigate chemically induced DNA damage. But in many cases liver tumors caused by indirect acting carcinogens can also originate from non-parenchymal liver cells (NPC). We used freshly isolated rat PC and NPC to demonstrate that only PC have activation capacity when treated in vitro with different genotoxic procarcinogens ( N-nitrosodimethylamine, NDMA; vinyl chloride, VC). The alkaline single cell microgel electrophoresis assay was applied to measure the genotoxic activity of the activated compounds. In order to test the hypothesis that reactive metabolites can be transported from PC to NPC, we performed additional in vivo studies as well as studies in which PC were incubated together with NPC, only separated by a dialysis tube (in vitro coincubation). The results indicate that reactive metabolites of both NDMA and VC are stable enough to be transported intercellularly from PC to NPC.

The single cell microgel electrophoresis assay (comet assay): technical aspects and applications. Report on the 5th LH Gray Trust Workshop, Institute of Cancer Research, 1994.

The single cell microgel electrophoresis assay (comet assay): technical aspects and applications. Report on the 5th LH Gray Trust Workshop, Institute of Cancer Research, 1994.