The in vitro transformation of a rat liver epithelial cell line with chromium

The transformation of a rat liver epithelial cell line under a wide range of doses of chromium was determined by anchorage-independent growth and tumor formation in syngeneic animals. Chronic exposure to low concentrations and brief exposure to high concentrations of hexavalent chromium (K₂CrO₄) transformed the cells, but one dose (1 mM K₂CrO₄, 2h) was clearly optimal in this regard. The cytotoxicity, effects on cell cycle, rates of chromium uptake, and mutagenic activity under the different treatment conditions were evaluated. The results showed that cells could adapt to the presence of chromium under certain treatment conditions, but this was not the case for the optimal transforming dose. Cells treated with chromium above the optimal transforming dose showed evidence of a transient G2 arrest, whereas all lower levels of treatment did not. A low level continuous exposure to chromate was mutagenic, whereas high level short exposures, including the optimal transforming dose, were not. An increase in the amount of protein complexed with isolated nucleic acids was detected in cells following treatment with the optimal transforming dose of chromate. The results indicate that the effects of chromium on this in vitro system vary with dose; and the identification of those events relevant to metal carcinogenesis will require consideration of treatment conditions.     

Gene expression changes in human lung cells exposed to arsenic, chromium, nickel or vanadium indicate the first steps in cancer

The complex process of carcinogenesis begins with transformation of a single cell to favor aberrant traits such as loss of contact inhibition and unregulated proliferation - features found in every cancer. Despite cancer's widespread prevalence, the early events that initiate cancer remain elusive, and without knowledge of these events cancer prevention is difficult. Here we show that exposure to As, Cr, Ni, or vanadium (V) promotes changes in gene expression that occur in conjunction with aberrant growth. We exposed immortalized human bronchial epithelial cells to one of four metals/metalloid for four to eight weeks and selected transformed clonal populations based upon anchorage independent growth of single cells in soft agar. We detected a metal-specific footprint of cancer-related gene expression that was consistent across multiple transformed clones. These gene expression changes persisted in the absence of the progenitor metal for numerous cell divisions. Our results show that even a brief exposure to a carcinogenic metal may cause many changes in gene expression in the exposed cells, and that from these many changes, the specific change(s) that each metal causes that initiate cancer likely arise.     

Effect of sodium butyrate on gene expression in a rat myogenic cell line

Sodium butyrate, when added in millimolar concentration to a culture of myoblasts of the L6 cell line, inhibits reversibly cell proliferation and differentiation. In the present work, we have studied the effect of Na butyrate on the translational efficiency of the overall poly (A)+ RNA. The mRNA from treated cells was translated in vitro as efficiently as proliferating myoblasts mRNA, while a decrease of translation efficiency was observed with myotubes mRNA. In addition this RNA directs the synthesis of several new polypeptides. on the switch on of alpha actin and myosin heavy chains (MHC), muscle specific genes by the dot blot and Northern blot techniques using cloned probes. Na butyrate prevented the expression of MHC and allowed the switch on of alpha actin gene but at a lesser extent than in normal myotubes. In addition the drug prevented the translocation of alpha actin mRNA into the cytoplasm.     

Curcumin causes promoter hypomethylation and increased expression of FANCF gene in SiHa cell line

Curcumin and resveratrol were evaluated for their potential to cause reversal of promoter hypermethylation and associated gene expression of FANCF in SiHa cell line. Methylation specific PCR along with bisulphite sequencing revealed the demethylation of 12 CpG sites out of 15 CpG sites spanning ?280 to ?432 region of FANCF promoter after treatment with curcumin and fivefold up regulation of FANCF gene expression as shown by qRT-PCR. In vitro methylation assay also showed that M.SssI an analogue of DNMT1 was effectively inhibited at 50 lM concentration of curcumin. Resveratrol was not found to be effective in causing reversal of promoter hypermethylation of FANCF gene when used at 20 lM for 4 days in SiHa cell line.     

Effects of long-term in vitro exposure to aluminum,cadmium or lead on differentiation and cholinergic receptor expression in a human neuroblastoma cell line

Neurotoxicity of long-term exposure to lead, aluminum and cadmium has been studied in vitro on the human neuroblastoma cell line IMR32 by measuring cytotoxicity, and the effects on neuronal-specific characteristics such as nitrite outgrowth and expression of cholinergic receptors as parameters of toxicity. Cytotoxicity was highest with cadmium, intermediate with lead and lowest with aluminum exposure. Lead, but not cadmium and aluminum, interfered with neurite growth. The expression of a-bungarotoxin binding sites and muscarinic receptors was markedly increased by cadmium and not affected by aluminum exposure. Lead induced only an increase of toxin binding sites. These in vitro modifications are discussed in relation to the possible use of neuronal cell lines for detecting neurotoxic effects of heavy metals.Abbreviations ACh acetylcholine - -Bgtx -bungarotoxin - BrdUr 5'-bromodeoxyuridine - CNS central nervous system     

Analysis of metal-induced mutations altering the expression or structure of a retroviral gene in a mammalian cell line

Carcinogenic metal compounds, with the exception of chromium(VI), have been found to be poorly mutagenic in both prokaryotic and mammalian cell mutagenesis assays, yet they are clearly clastogenic (Hansen and Stern, 1984). Thus, the role of metals as initiators in carcinogenesis has been difficult to delineate. In an effort to develop a model system capable of assaying DNA damage caused by carcinogenic metals, we have investigated the role of NiCl2, CdCl2, Na2CrO4, and NMU in a murine sarcoma virus-infected mammalian cell line in which expression of the retroviral v-mos gene is growth-temperature regulated. This cell line, designated 6m2, contains a single-copy, stably integrated, mutant Moloney murine sarcoma virus DNA (designated MuSVts110) and is temperature sensitive for morphological transformation due to a conditionally defective viral RNA-splicing event that in turn regulates expression of the viral transforming gene. Mutations affecting the viral DNA in 6m2 cells can be detected if these alterations lead to changes in the structure or expression of the transforming protein encoded by the MuSVts110 v-mos gene. Analysis of the viral proteins from 6m2 'revertant' cell lines (as defined by reversion to the transformed phenotype at all growth temperatures) selected after treatment with the above agents showed that NiCl2, NMU, and Na2CrO4 each induced a different yet specific type of mutation. NiCl2 and NMU each altered the temperature sensitivity of viral RNA splicing, possibly due to base substitution mutations, but did so to distinctly different extents. Na2CrO4 affected the structure of the viral proteins by inducing what appear to be short frameshift mutations that resulted in the temperature-dependent translation of a novel virus-encoded transforming protein, P100gag-mos. CdCl2 also induced frameshift mutations but, in one case, induced a mutation which may result from a deletion of about 300 bases within the MuSVts110 DNA.     

Cell surface changes in a cell line derived from a human pleural effusion

The phenomenon of blebbing has been recorded in cells obtained from a pleural effusion of a patient with a small cell lung carcinoma. The blebbing manifested itself as a sudden ballooning of the cell membrane which then moved around the perimeter of the cell as a sausage-shaped swelling. This was followed by its rapid collapse. The rapidity of the process allowed real time video recordings, not time lapse, and still frames from the recording were photographed. Cells were also viewed using a scanning electron microscope and more detail of the blebs was revealed. Possible functions of cell blebbing are discussed.     

Exposure of developing oligodendrocytes to cadmium causes HSP72 induction, free radical generation, reduction in glutathione levels, and cell death

Primary cultures of oligodendrocytes were used to study the toxic effects of cadmium chloride. Cell viability was evaluated by the mitochondrial dehydrogenase activity and confirmed by propidium iodide (PI) fluorescence staining. The expression of the 72 kDa stress protein, HSP72, was assayed by Western blot analysis. The results showed that Cd(2+)-induced toxicity was dependent on the time and dose of exposure, as well as on the developmental stage of the cultures. Oligodendrocyte progenitors were more vulnerable to Cd(2+) toxicity than were mature oligodendrocytes. Mature oligodendrocytes accumulated relatively higher levels of Cd(2+) than did progenitors, as determined by (109)CdCl(2) uptake; treatment with the metal ion caused a more pronounced reduction in intracellular glutathione levels and significantly higher free radical accumulation in progenitors. The latter could explain the observed differences in Cd(2+) susceptibility. HSP72 protein expression was increased both in progenitors and in mature cells exposed to Cd(2+). Pretreatment with N-acetylcysteine, a thiocompound with antioxidant activity and a precursor of glutathione, prevented Cd(2+)-induced (i) reduction in glutathione levels and (ii) induction of HSP72 and diminished (i) Cd(2+) uptake and (ii) Cd(2+)-evoked cell death. In contrast, buthionine sulfoximine, an inhibitor of gamma-glutamyl-cysteine synthetase, depleted glutathione, and potentiated the toxic effect of Cd(2+). These results strongly suggest that Cd(2+)-induced cytotoxicity in oligodendrocytes is mediated by reactive oxygen species and is modulated by glutathione levels.     

Fear conditioning induces distinct patterns of gene expression in lateral amygdala

The lateral nucleus of the amygdala (LA) has been implicated in the formation of long-term associative memory (LTM) of stimuli associated with danger through fear conditioning. The current study aims to detect genes that are expressed in LA following associative fear conditioning. Using oligonucleotide microarrays, we monitored gene expression in rats subjected to paired training where a tone co-terminates with a footshock, or unpaired training where the tone and footshock are presented in a non-overlapping manner. The paired protocol consistently leads to auditory fear conditioning memory formation, whereas the unpaired protocol does not. When the paired group was compared with the unpaired group 5 h after training, the expression of genes coding for the limbic system-associated membrane protein (Lsamp), kinesin heavy chain member 2 (Kif2), N -ethylmaleimide-sensitive fusion protein (NSF) and Hippocalcin-like 4 protein (Hpcal4) was higher in the paired group. These genes encode proteins that regulate neuronal axonal morphology (Lsamp, Kif2), presynaptic vesicle cycling and release (Hpcal4 and NSF), and AMPA receptor maintenance in synapses (NSF). Quantitative real-time PCR (qPCR) showed that Kif2 and Lsamp are expressed hours following fear conditioning but minutes after unpaired training. Hpcal4 is induced by paired stimulation only 5 h after the training. These results show that fear conditioning induces a unique temporal activation of molecular pathways involved in regulating synaptic transmission and axonal morphology in LA, which is different from non-associative stimulation.     

Identification of depot-specific human fat cell progenitors through distinct expression profiles and developmental gene patterns

Anatomically separate fat depots differ in size, function, and contribution to pathological states, such as the metabolic syndrome. We isolated preadipocytes from different human fat depots to determine whether the basis for this variation is partly attributable to differences in inherent properties of fat cell progenitors. We found that genome-wide expression profiles of primary preadipocytes cultured in parallel from abdominal subcutaneous, mesenteric, and omental fat depots were distinct. Interestingly, visceral fat was not homogeneous. Preadipocytes from one of the two main visceral depots, mesenteric fat, had an expression profile closer to that of subcutaneous than omental preadipocytes, the other main visceral depot. Expression of genes that regulate early development, including homeotic genes, differed extensively among undifferentiated preadipocytes isolated from different fat depots. These profiles were confirmed by real-time PCR analysis of preadipocytes from additional lean and obese male and female subjects. We made preadipocyte strains from single abdominal subcutaneous and omental preadipocytes by expressing telomerase. Depot-specific developmental gene expression profiles persisted for 40 population doublings in these strains. Thus, human fat cell progenitors from different regions are effectively distinct, consistent with different fat depots being separate mini-organs.     

Toxicogenomic analysis of gene expression changes in rat liver after a 28-day oral benzene exposure

Benzene is an industrial chemical, component of automobile exhaust and cigarette smoke. After hepatic bioactivation benzene induces bone marrow, blood and hepatic toxicity. Using a toxicogenomics approach this study analysed the effects of benzene at three dose levels on gene expression in the liver after 28 daily doses. NMR based metabolomics was used to assess benzene exposure by identification of characteristic benzene metabolite profiles in urine. The 28-day oral exposure to 200 and 800 mg/kg/day but not 10 mg/kg/day benzene-induced hematotoxicity in male Fisher rats. Additionally these upper dose levels slightly reduced body weight and increased relative liver weights. Changes in hepatic gene expression were identified with oligonucleotide microarrays at all dose levels including the 10 mg/kg/day dose level where no toxicity was detected by other methods. The benzene-induced gene expression changes were related to pathways of biotransformation, glutathione synthesis, fatty acid and cholesterol metabolism and others. Some of the effects on gene expression observed here have previously been observed after induction of acute hepatic necrosis with bromobenzene and acetaminophen. In conclusion, changes in hepatic gene expression were found after treatment with benzene both at the toxic and non-toxic doses. The results from this study show that toxicogenomics identified hepatic effects of benzene exposure possibly related to toxicity. The findings aid to interpret the relevance of hepatic gene expression changes in response to exposure to xenobiotics. In addition, the results have the potential to inform on the mechanisms of response to benzene exposure.     

Exposure of rat brain to 915 MHz GSM microwaves induces changes in gene expression but not double stranded DNA breaks or effects on chromatin conformation

We investigated whether exposure of rat brain to microwaves (MWs) of global system for mobile communication (GSM) induces DNA breaks, changes in chromatin conformation and in gene expression. An exposure installation was used based on a test mobile phone employing a GSM signal at 915 MHz, all standard modulations included, output power level in pulses 2 W, specific absorption rate (SAR) 0.4 mW/g. Rats were exposed or sham exposed to MWs during 2 h. After exposure, cell suspensions were prepared from brain samples, as well as from spleen and thymus. For analysis of gene expression patterns, total RNA was extracted from cerebellum. Changes in chromatin conformation, which are indicative of stress response and genotoxic effects, were measured by the method of anomalous viscosity time dependencies (AVTD). DNA double strand breaks (DSBs) were analyzed by pulsed-field gel electrophoresis (PFGE). Effects of MW exposure were observed on neither conformation of chromatin nor DNA DSBs. Gene expression profiles were obtained by Affymetrix U34 GeneChips representing 8800 rat genes and analyzed with the Affymetrix Microarray Suite (MAS) 5.0 software. In cerebellum from all exposed animals, 11 genes were upregulated in a range of 1.34-2.74 fold and one gene was downregulated 0.48-fold (P < .0025). The induced genes encode proteins with diverse functions including neurotransmitter regulation, blood-brain barrier (BBB), and melatonin production. The data shows that GSM MWs at 915 MHz did not induce PFGE-detectable DNA double stranded breaks or changes in chromatin conformation, but affected expression of genes in rat brain cells.