Intracellular measurements in mammary carcinoma cells using fiber-optic nanosensors

Submicrometer fiber-optic biosensors have been developed and used to measure toxic chemicals within single cells. Optical fibers that have been pulled to a distal-end diameter of less than 1 micrometer are coated with antibodies to selectively bind the species of interest. This paper describes the use of these fibers to selectively measure the concentration of benzo[a]pyrene tetrol (BPT), a metabolite of benzo[a]pyrene, within individual cells of two different cell lines, human mammary carcinoma cells and rat liver epithelial cells. The results from these measurements have been used to determine the sensitivity, reproducibility, and usefulness of these nanosensors. The detection limit of these biosensors has been determined to be 0.64 +/- 0.17 x 10(-11) M for BPT.     

Effects of pretreatment with benzo[a]pyrene on the stereochemical selectivity of metabolic activation of benzo[a]pyrene to DNA-binding metabolites in hamster embryo cell cultures

The proportions of individual benzo[a]pyrene (BaP)-DNA adducts present in rodent embryo cell cultures change with the length of time of exposure to BaP; the major alteration is an increase in the proportion of (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydroBaP (BaPDE)-deoxyguanosine (dG) adduct (Sebti et al., Cancer Res., 45 (1984) 1594-1600). To determine if this change in the BaP-DNA adducts could result from the induction of enzymes involved in oxidation of BaP, hamster embryo cell cultures were exposed to acetone or BaP for 24 h and then the medium was replaced with fresh medium containing [3H]BaP. After 5 h the BaP-pretreated cells had a 30% higher level of binding of BaP to DNA and formed a greater proportion of (+)-anti-BaPE-dG adduct than the acetone-pretreated control group. Cells pretreated for 24 h with BaP and then exposed to [3H]BaP and Actinomycin D for 5 h had a lower level of binding of BaP to DNA and a lower amount of (+)-anti-BaPDE-deoxyguanosine adduct than cells pretreated with acetone and exposed to [3H]BaP for 5 h. In contrast, pretreatment for 24 h with BaP plus Actinomycin D followed by a 5-h exposure to [3H]BaP resulted in a decrease in overall binding of BaP to DNA but had no effect on the amount of (+)-anti-BaPDE-deoxyguanosine adduct. Actinomycin D treatment had no significant effect on either the total amount of BaP metabolized, the formation of primary and water-soluble BaP metabolites, or cell viability, but reduced [3H]uridine incorporation into RNA by more than 65% at all times. These results suggest that induction of specific isozymes of cytochrome P-450 may be involved in the time-dependent increase in the proportion of (+)-anti-BaPDE-DNA adducts in BaP-treated cells. The state of induction of specific isozymes of cytochrome P-450 and the ability of the BaP dose applied to induce them may be major factors in determining the proportion of BaP metabolized to (+)-anti-BaPDE, the most carcinogenic stereoisomer of BaPDE.     

Inducible cytochrome P450 activities in renal glomerular mesangial cells: biochemical basis for antagonistic interactions among nephrocarcinogenic polycyclic aromatic hydrocarbons

BACKGROUND: Benzo(a)pyrene (BaP), anthracene (ANTH) and chrysene (CHRY) are polynuclear aromatic hydrocarbons (PAHs) implicated in renal toxicity and carcinogenesis. These PAHs elicit cell type-specific effects that help predict toxicity outcomes in vitro and in vivo. While BaP and ANTH selectively injure glomerular mesangial cells, and CHRY targets cortico-tubular epithelial cells, binary or ternary mixtures of these hydrocarbons markedly reduce the overall cytotoxic potential of individual hydrocarbons. METHODS: To study the biochemical basis of these antagonistic interactions, renal glomerular mesangial cells were challenged with BaP alone (0.03 - 30 microM) or in the presence of ANTH (3 microM) or CHRY (3 microM) for 24 hr. Total RNA and protein will be harvested for Northern analysis and measurements of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin-O-deethylase (EROD) activity, respectively, to evaluate cytochrome P450 mRNA and protein inducibility. Cellular hydrocarbon uptake and metabolic profiles of PAHs were analyzed by high performance liquid chromatography (HPLC). RESULTS: Combined hydrocarbon treatments did not influence the cellular uptake of individual hydrocarbons. ANTH or CHRY strongly repressed BaP-inducible cytochrome P450 mRNA and protein expression, and markedly inhibited oxidative BaP metabolism. CONCLUSION: These findings indicate that antagonistic interactions among nephrocarcinogenic PAHs involve altered expression of cytochrome P450s that modulate bioactivation profiles and nephrotoxic/ nephrocarcinogenic potential.     

On-line monitoring of cell concentration of Perilla frutescens in a bioreactor

This article demonstrates the successful in situ real-time monitoring of the cell concentration of Perilla frutescens in a bioreactor by using a laser turbidimeter. It was found that turbidity measurements at 780 nm with the laser sensor were hardly affected by the red color of the anthocyanin produced by P. frutescens cells, nor by the aeration rate or agitation speed within the ranges investigated. There was an excellent linear relationship, with a correlation coefficient (r(2)) higher than 0.99, between the sensor's response and the cell concentration. The whole growth stage of the cells, i.e., lag, logarithmic, and stationary phases, in bioreactor cultivations, could be satisfactorily estimated on-line by means of the in situ turbidimeter. However, during the declining phase of the cells, an apparent deviation was observed between the on-line estimations and off-line measurements of cell concentrations by dry cell weight, while the wet cell weight could be estimated by the same turbidimeter system. We found that this deviation was caused by a decrease in the cell density due to an increase of the individual cell volume and a decrease of the cell dry weight during the declining phase. (c) 1993 John Wiley & Sons, Inc.     

Recognition of individual genes in a single bacterial cell by fluorescence in situ hybridization--RING-FISH

Fluorescence in situ hybridization (FISH) using rRNA targeted oligonucleotide probes is a standard method for identification of microorganisms in environmental samples. Apart from its value as a phylogenetic marker ribosomal RNA has always been the favoured target molecule for FISH because of its abundance in all cells, whereas plasmids and DNA were regarded as unsuitable targets because of their low copy number. Here we present an improved FISH technique, which is based on polynucleotide probes. It goes beyond the detection of high copy intracellular nucleic acids such as rRNA (up to 10(4)-10(5) copies per cell) and allows for the first time the in situ detection of individual genes or gene fragments on plasmids (10(1)-10(3) copies per cell) and chromosomal DNA (<10 copies per cell) in a single cell. Using E. coli as model organism we were able to detect in situ cells harbouring the antibiotic resistance gene beta lactamase on high, medium and low copy plasmids as well as the chromosomal encoded housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Furthermore, we detected the prepilin peptidase gene xpsO in the plant pathogen Xanthomonas campestris in situ. Because of the characteristic hybridization signal obtained with this method--a halo-like, ring-shaped concentration of fluorescence in the cell periphery--we coined the term RING-FISH (recognition of individual genes) to differentiate it from conventional FISH.     

Benzo(a)pyrene inhibits endometrial cell apoptosis in early pregnant mice via the WNT5A pathway

Benzo(a)pyrene (BaP) is an endocrine-disrupting pollutant present in various aspects of daily life, and studies have demonstrated that BaP exerts reproductive toxicity. We previously showed that BaP damages endometrial morphology and decreases the number of implantation sites in early pregnant mice, but the mechanisms underlying these effects remain unclear. The endometrial function is crucial for implantation, which is associated with endometrial cell apoptosis. In this study, we focused on the effect of BaP on endometrial cell apoptosis and the role of WNT signaling during this process. Pregnant mice were gavaged with corn oil (control group) or 0.2 mg·kg⁻¹·day ⁻¹ BaP (treatment group) from Days 1 to 6 of pregnancy. BaP impaired endometrial function by decreasing the expression of HOXA10 and BMP2, two markers of receptivity and decidualization. WNT5A and β-catenin were activated in the BaP group. BaP affected the expression of apoptosis-related proteins and inhibited the apoptosis of endometrial stromal cells. In vitro, human endometrial stromal cells (HESCs) were treated with different concentrations of BaP (dimethyl sulfoxide (DMSO); 5, 10 µM). WNT5A and β-catenin were also upregulated in the BaP treatment group. HESC apoptosis was restrained by BaP. Inhibiting WNT5A by SFRP5 partially restored the effect of BaP on apoptosis. In summary, these results suggested that BaP exposure during early pregnancy activates WNT5A/β-catenin signaling pathway, which inhibits the endometrial cell apoptosis and potentially destroys endometrial function.     

Haploid vegetative mycelia of Armillaria gallica show among-cell-line variation for growth and phenotypic plasticity

Vegetative mycelial cells of Armillaria are expected to have diploid nuclei. Cells from a single mycelium therefore would not be expected to differ from one another for ecologically relevant quantitative traits. We isolated two sets of basidiome cell lines (from spores and stipe cells) and one set of vegetative cell lines (from an attached rhizomorph) from a single contiguous Armillaria gallica mycelium. We isolated a second set of vegetative cell lines from the soil 20 cm from the above basidiome-rhizomorph complex. In all four sets of cell lines in situ DAPI-DNA measurements showed cells are haploid and quantitative-trait analyses of cell lines grown at different water potentials revealed high levels of among-cell-line genetic variation for both growth and phenotypic plasticity. Haploidy and the existence of ecologically relevant genetic variation within vegetative individuals are unexpected and mean that a process similar to evolutionary adaptation could take place within the soma of a genetic individual. We believe this is a key to understanding how large A. gallica mycelia survive exposure to variation in ecological conditions during lives that potentially span several tree (host) generations.     

Co-exposure to benzo[a]pyrene and UVA induces phosphorylation of histone H2AX

Phosphorylation of histone H2AX (termed gamma-H2AX) was recently identified as an early event after induction of DNA double strand breaks (DSBs). We have previously shown that co-exposure to benzo[a]pyrene (BaP), a wide-spread environmental carcinogen, and ultraviolet A (UVA), a major component of solar UV radiation, induced DSBs in mammalian cells. In the present study, we examined whether co-exposure to BaP and UVA generates gamma-H2AX in CHO-K1 cells. Single treatment with BaP (10(-9)-10(-7)M) or UVA ( approximately 2.4 J/cm(2)) did not result in gamma-H2AX, however, co-exposure drastically induced foci of gamma-H2AX in a dose-dependent manner. gamma-H2AX could be detected even at very low concentration of BaP (10(-9)M) plus UVA (0.6J/cm(2)), which did not change cell survival rates. NaN(3) effectively inhibited the formation of gamma-H2AX induced by co-exposure, indicating the contribution of singlet oxygen. This is the first evidence that co-exposure to BaP and UVA induced DSBs, involving gamma-H2AX.     

mRNA detection of individual cells with the single cell nanoprobe method compared with in situ hybridization

Background  

The localization of specific mRNA generates cell polarity by controlling the translation sites of specific proteins. Although most of these events depend on differences in gene expression, no method is available to examine time dependent gene expression of individual living cells. In situ hybridization (ISH) is a powerful and useful method for detecting the localization of mRNAs, but it does not allow a time dependent analysis of mRNA expression in single living cells because the cells have to be fixed for mRNA detection. To overcome these issues, the extraction of biomolecules such as mRNAs, proteins, and lipids from living cells should be performed without severe damage to the cells. In previous studies, we have reported a single cell nanoprobe (SCN) method to examine gene expression of individual living cells using atomic force microscopy (AFM) without killing the cells.     

Characterization of genomic single nucleotide polymorphism via colorimetric detection using a single gold nanoprobe

Identification of specific nucleic acid sequences mediated by gold nanoparticles derivatized thiol-modified oligonucleotides (Au–nanoprobes) has been proven to be a useful tool in molecular diagnostics. Here, we demonstrate that, on optimization, detection may be simplified via the use of a single Au–nanoprobe to detect a single nucleotide polymorphism (SNP) in homo- or heterozygote condition. We validated this non-cross-linking approach through the analysis of 20 clinical samples using a single specific Au–nanoprobe for an SNP in the FTO (fat mass and obesity-associated) gene against direct DNA sequencing. Sensitivity, specificity, and limit of detection (LOD) were determined, and statistical differences were calculated by one-way analysis of variance (ANOVA) and a post hoc Tukey’s test to ascertain whether there were any differences between Au–nanoprobe genotyped groups. For the first time, we show that the use of a single Au–nanoprobe can detect SNP for each genetic status (wild type, heterozygous, or mutant) with high degrees of sensitivity (87.50%) and specificity (91.67%).     

Role of poly(ADP-ribose) glycohydrolase silencing in DNA hypomethylation induced by benzo(a)pyrene

Benzo(a)pyrene (BaP) is a known carcinogen cytotoxic which can trigger extensive cellular responses. Many evidences suggest that inhibitors of poly(ADP-ribose) glycohydrolase (PARG) are potent anticancer drug candidates. However, the role of PARG in BaP carcinogenesis is less understood. Here we used PARG-deficient human bronchial epithelial cell line (shPARG cell) as an in vitro model, and investigated the role of PARG silencing in DNA methylation pattern changed by BaP. Our study shows, BaP treatment decreased global DNA methylation levels in 16HBE cells in a dose-dependent manner, but no dramatic changes were observed in shPARG cells. Further investigation revealed PARG silencing protected DNA methyltransferases (DNMTs) activity from change by BaP exposure. Interestingly, Dnmt1 is PARylated in PARG-null cells after BaP exposure. The results show a role for PARG silencing in DNA hypomethylation induced by BaP that may provide new clue for cancer therapy.     

Optimization of conditions for in situ hybridization and determination of the relative number of ribosomes in the cells of the mature embryo sac of maize

Summary We have initiated experiments to understand the molecular regulation of embryo sac development in flowering plants by a study of ribosome synthesis and accumulation. Because of the very small size of the embryo sac and the large volume of ovule tissue it is embedded in, in situ hybridization with nucleic acid probes is presently the only practical method for such molecular measurements on individual cells of the embryo sac. Methods of tissue preparation, sectioning and screening of ovules for embryo sac containing sections, in situ hybridization using a ribosomal mRNA probe, and staining were optimized. Relative densities of silver grains for individual cells of the mature maize (W22) embryo sac were determined from in situ hybridizations. The silver grain counts are directly related to the numbers of ribosomes. Volumes of individual cells were determined by confocal microscope image analysis, and this permitted the calculation of the relative total numbers of ribosomes in individual cells of the embryo sac and nucellus. The central cell has a volume 260 times that of a nucellar cell at the micropylar end of the ovule, 15 times that of the egg cell, 30 times that of a synergid, and 130 times the volume of an antipodal cell. The mature maize embryo sac has 20 or more antipodal cells. The central cell has approximately 200 times the number of ribosomes as are present in a nucellar cell, about 7 times as many ribosomes as are in the egg cell, 14 times as many ribosomes as in each synergid, and about 80 times the ribosome content of individual antipodal cells. The data are discussed with respect to the utilization of the ribosomes following fertilization in the early embryo and endosperm.     

Endogenous assays of DNA methyltransferases: Evidence for differential activities of DNMT1, DNMT2, and DNMT3 in mammalian cells in vivo

While CpG methylation can be readily analyzed at the DNA sequence level in wild-type and mutant cells, the actual DNA (cytosine-5) methyltransferases (DNMTs) responsible for in vivo methylation on genomic DNA are less tractable. We used an antibody-based method to identify specific endogenous DNMTs (DNMT1, DNMT1b, DNMT2, DNMT3a, and DNMT3b) that stably and selectively bind to genomic DNA containing 5-aza-2'-deoxycytidine (aza-dC) in vivo. Selective binding to aza-dC-containing DNA suggests that the engaged DNMT is catalytically active in the cell. DNMT1b is a splice variant of the predominant maintenance activity DNMT1, while DNMT2 is a well-conserved protein with homologs in plants, yeast, Drosophila, humans, and mice. Despite the presence of motifs essential for transmethylation activity, catalytic activity of DNMT2 has never been reported. The data here suggest that DNMT2 is active in vivo when the endogenous genome is the target, both in human and mouse cell lines. We quantified relative global genomic activity of DNMT1, -2, -3a, and -3b in a mouse teratocarcinoma cell line. DNMT1 and -3b displayed the greatest in vivo binding avidity for aza-dC-containing genomic DNA in these cells. This study demonstrates that individual DNMTs can be tracked and that their binding to genomic DNA can be quantified in mammalian cells in vivo. The different DNMTs display a wide spectrum of genomic DNA-directed activity. The use of an antibody-based tracking method will allow specific DNMTs and their DNA targets to be recovered and analyzed in a physiological setting in chromatin.     

Influence of long term treatment with 3-methylcholanthrene or carbaryl on mixed-function oxidase activity in 3T3 fibroblasts: Studies by microspectrofluorimetry on single cells

Using benzo(a)pyrene (BaP) as a probe for aryl hydrocarbon hydroxylase (AHH) activity, differences in mixed-function oxidase (MFO) activity were observed using microspectrofluorimetry in single living cells during long term treatment with 3-methylcholanthrene (3-MC) or carbaryl. Although these two compounds differ in chemical structure, similar effects were observed in 3T3 cell populations. The results suggest that the two compounds activate the same enzymatic system and that individual cells of a supposed homogeneous cell population are not equally sensitive to xenobiotics, i.e. subpopulations were observed which have differences in AHH activity.     

Neuroendocrine epithelial cells in the broncho-parabronchial transition of embryonic, immature and mature quail (Coturnix coturnix)

The main objective of this integrated light microscopic, transmission and scanning electron microscopic study was to describe in greater detail the structural pattern and developmental stages of pulmonary neuroendocrine epithelial cells (PNECs) in the broncho-parabronchial transition (BPT) of both developing and mature quail. In mature quail the BPT appeared as a diaphragm opening into the parabronchial vestibulum. Perpendicular sections revealed two bilayered crest-like entrance folds invested by a uniform population of granular cells with lamellar bodies and a brush border of blunt microvilli. Solitary PNECs were found interlaced between the granular cells on both sides of the BPT. In addition, PNECs with small dense-cored vesicles (DCVs) were found subepithelially in the lamina propria mucosae of the parabronchial compartment of the BPT, which was surrounded by a capsule of granular cells. Furthermore, clusters of PNECs devoid of any epithelial capsule, but with large DCVs were located in the tunica propria mucosae. The first signs of the developing BPT could be identified as early as embryonic day (ED) 13. By ED 17, the area of the future BPT was seen to be invested by a uniform population of granular cells but the entrance fold proper did not appear until after hatching. Solitary or clustered PNECs were demonstrated in parabronchial buds growing into the mesenchyme on ED 12 and 13: either type I PNECs with small DCVs measuring about 80-120 nm or type II PNECs with large dense granules measuring approximately 150-280 nm. Both types of PNECs represented a temporary cell population in the prehatching period. In the parabronchus no PNECs or neuroendocrine epithelial bodies could be demonstrated in mature and immature quail beyond the BPT.     

Benzo(a)pyrene,but not 2,3,7,8-tetrachlorodibenzo-p-dioxin,alters cell adhesion proteins in human uterine RL95-2 cells

This study compared the effects of benzo(a)pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), two aryl hydrocarbon receptor agonists, on cell attachment and adherens junction proteins in RL95-2 human uterine endometrial cells. Exposure to 10 microM BaP significantly decreased cell attachment to Matrigel, whereas 10 nM TCDD had no effect. Immunocytochemistry and Western immunoblot analysis showed that BaP, but not TCDD, produced a marked loss of plasma membrane epidermal growth factor receptor (EGF-R) localized along intercellular boundaries. BaP-treated cells exhibited significant decreases in beta-catenin and cadherin protein levels, while vinculin levels remained unchanged relative to control. In contrast, TCDD treatment had no effect on the levels of beta-catenin, cadherin, or vinculin. Further studies using the fluorescein labeled peptide phalloidin showed the presence of continuous subcortical actin filaments in control cells, whereas BaP-treated cells had subcortical actin aggregates. Thus, in contrast to TCDD, BaP produces a loss of cell attachment involving decreased localization of molecules important for cell-cell interactions in RL95-2 cells.     

Comparison of the effects of arsenic and cadmium on benzo(a)pyrene-induced micronuclei in mouse bone-marrow

This study was undertaken to investigate the genotoxic interactions between the common environmental pollutants: arsenic (As), cadmium (Cd) and benzo(a)pyrene (BaP), which are known to be human carcinogens. C57BL/6J/Han mice were pre-treated with 100mg cadmium chloride (Cd(2+))/L or 50mg sodium arsenite (As(3+))/L in drinking water for 7 days and then given a single dose of 200mg BaP/kg bw by intra-peritoneal injection. A third group of mice did not receive the pre-treatment and was given BaP alone. Mice were sacrificed before or at 12, 24, 48 or 72h after BaP administration. Chromosome damage in bone-marrow cells was assessed by use of the micronucleus test. The study revealed that BaP induced a statistically significant increase in micronucleus (MN) frequency at 48h after administration. In animals exposed to Cd in drinking water no enhancement of genotoxicity was observed compared with the control group that was given tap water only. In Cd/BaP co-exposed animals, the MN frequency at respective time points did not differ from that for the animals exposed solely to BaP. A statistically higher MN frequency was found in bone marrow of animals exposed to As compared with controls that received tap water (0.92+/-0.29% versus 0.38+/-0.13%, respectively). This effect was even more pronounced after combined exposure to As and BaP. In the co-exposed animals, significantly elevated levels of MN were detected in samples examined at 12, 24 and 48h after BaP administration, compared with animals receiving BaP alone (1.14+/-0.31%, 1.26+/-0.3% and 2.02+/-0.45% versus 0.44+/-0.13%, 0.44+/-0.11% and 1.04+/-0.44%, respectively). These findings imply strong interactions between As and BaP, but not between Cd and BaP, in inducing DNA damage in polychromatic erythrocytes in mouse bone-marrow.     

Effect of PARP-1 deficiency on DNA damage and repair in human bronchial epithelial cells exposed to Benzo(a)pyrene

Benzo[a]pyrene is a ubiquitously distributed environmental pollutant known to cause DNA damage, whereas PARP-1 is a nuclear enzyme that is activated by damaged DNA and plays an important role in base excision repair and genomic stability. Here, 16HBE and its PAPR1-deficient cells were exposed to BaP, and the DNA damage level and repair ability of both cell lines were measured by alkaline comet assay. The results showed that cell viability of both cell lines decreased in a dose-dependent manner when exposed to BaP, but there was no significant difference between two cell lines. Comet assay showed that BaP caused DNA damage in both cell lines at an obvious dose- and time-dependent manner. Compare with 16HBE, the PARP1-deficient cells were more sensitive to the damage caused by BaP. The results of DNA repair experiment showed that both cell lines can recover from the damage in a time-dependent pattern. The relative repair percentage of PARP1-deficient cells were generally lower than that of 16HBE at all exposed concentrations at the early stage of repair, but tended to be closer between two cell lines at the later period. According to results, we came to the conclusion that PARP1-deficient cells were more sensitive to BaP in contrast to normal 16HBE; DNA repair capacity in PARP1-deficient cells decreased significantly at the early stage of repair, but increased to the equivalent level of normal 16HBE in the later period. PARP-1 plays an important role in early repair of DNA damage caused by BaP in 16HBE notwithstanding the main repair work is taken by NER pathway.