Dimethylsulfoxide induces aneuploidy in a fungal test system

Summary Errors in chromosome segregation leading to numerical anomalies appear to be unusually frequent in Man and consequently a large proportion of conceptions in our species are aneuploid. Concer has been expressed that this frequency may be increased still further following exposure to inducing substances (trisomigens) present in the environment. We have been developing a fungal test system to screen for such trisomigens and in this paper we report its use in detecting induction following exposure to dimethylsulfoxide (DMSO). In our system DMSO induces segregational errors at both the first and second meiotic division. The results also show that increases in aneuploidy are proportional to the underlying spontaneous frequency. If this finding is generally true it will be especially important to avoid exposure to trisomigens as Man might be especially vulnerable to them.     

Current status of aneuploidy testing in Drosophila

Based on the literature on file at EMIC, 181 papers contained material on aneuploidy testing. Initial screening rejected papers providing no data, no negative control and/or poorly designed genetic schemes; 67 papers representing tests of 76 compounds were reported on. Statistical classifications were established as follows: (+)=a statistically significant difference at the 5% level between the treated and control frequencies; (-)=no significant difference at the 5% level when the number of offspring tested was sufficient to have identified an increase of 0.2% over the control with a power of 75%; I=inconclusive= (a) no significant difference at the 5% level but the number of offspring tested was below that necessary to detect an increase of 0.2% with a power of 75%; (b) the nature of apparent complete loss is undetermined; or (c) the nature of the germ cells sampled is not determined. Of the 76 compounds analyzed, calls were made on 34 compounds. 17/34 compounds were positive for chromosome gain (11/34 for chromosome gain and chromosome loss, 6/34 for chromosome gain only). 17/34 compounds were negative for chromosome gain (11/34 for chromosome gain and chromosome loss and 6 for chromosome gain only). Are any fo the compounds found to induce aneuploidy specific for aneuploid induction? 7 or the compounds positive for chromosome gain were positive in one or more tests assaying for other genetic endpoints, and no reliable data exists regarding results in other tests for the remaining 10 compounds; accordingly, the answer to the question awaits further work.     

Rotenone induces aneuploidy, polyploidy and endoreduplication in cultured Chinese hamster cells

The clastogenic potential of rotenone, an insecticide, was investigated in cultured Chinese hamster cells. Rotenone induced aneuploidy (hypodiploidy and hyperdiploidy), polyploidy, and endoreduplication, but not structural chromosome aberrations. The highest frequency of polyploidy and endoreduplication was 58.8% and 3.0%, respectively, when cells were treated with rotenone at 1.0 microgram/ml for 30 h.     

Polyploidy and aneuploidy induced by colcemid in Drosophila melanogaster

Colcemid was fed to Drosophila melanogaster larvae throughout most of the larval period. Surviving individuals were then mated with untreated flies, and their progeny were examined for polyploid flies or flies resulting from X-chromosome nondisjunction. A total of 251 polyploid offspring was recovered from the experimental matings, none from the control. All of the polyploids were evidently triploids, and all but one were obtained from colcemid-fed females: males produced significantly lower frequencies of triploid offspring than females. The highest average frequency of triploid offspring obtained from any treatment group was 18%. Nonrandom distributions of triploid offspring were observed among females raised identically, indicating tht polyploidization occurs mitotically, rather than meiotically, giving rise to clones of tetraploid oogonia. 9 colcemid-fed females produced exclusively triploid offspring. Colcemid also caused a significant increase in X-chromosome nondisjunction in females, though the frequencies of such offspring were at least several-fold lower than the frequencies of triploid offspring. Somatic polyploidy was apparently also indiced since patches of large cells were found on the wings of some flies raised on colcemid-containing food. Various teratological abnormalities were observed among the treated flies, including deformed or missing eyes and partially duplicated thoraxes.     

Chrysotile and amosite asbestos induce germ-line aneuploidy in Drosophila

Asbestos toxicity is a problem of considerable public concern and debate, however little is known regarding the biological targets of asbestos fibers. Prompted by reports that asbestos induces aneuploidy in cultured mammalian cells, we have investigated whether asbestos induces germ-line aneuploidy in Drosophila melanogaster. Using the ZESTE genetic test system, we have shown that both chrysotile and amosite asbestos induce sex-chromosome aneuploidy in Drosophila oocytes. Chrysotile appeared to be the more effective agent because it induced approximately equal frequencies of chromosome gain and chromosome loss, while amosite induced chromosome loss only. Two other asbestiform minerals, crocidolite and tremolite, were ineffective in this assay system. These results suggest that possible germ-line effects of asbestos should be considered in evaluating its potential impact on human health.     

LKB1 induces apical trafficking of Silnoon, a monocarboxylate transporter, in Drosophila melanogaster

Silnoon (Sln) is a monocarboxylate transporter (MCT) that mediates active transport of metabolic monocarboxylates such as butyrate and lactate. Here, we identify Sln as a novel LKB1-interacting protein using Drosophila melanogaster genetic modifier screening. Sln expression does not affect cell cycle progression or cell size but specifically enhances LKB1-dependent apoptosis and tissue size reduction. Conversely, down-regulation of Sln suppresses LKB1-dependent apoptosis, implicating Sln as a downstream mediator of LKB1. The kinase activity of LKB1 induces apical trafficking of Sln in polarized cells, and LKB1-dependent Sln trafficking is crucial for triggering apoptosis induced by extracellular butyrate. Given that LKB1 functions to control both epithelial polarity and cell death, we propose Sln is an important downstream target of LKB1.     

Aneuploidy in Drosophila, IV. Inhalation studies on the induction of aneuploidy by nitriles

The Drosophila ZESTE system was used to monitor the induction of sex chromosome aneuploidy following inhalation exposure of adult females to four nitriles: acetonitrile, propionitrile, acrylonitrile and fumaronitrile. Acetonitrile and propionitrile were highly effective aneuploidogens, inducing both chromosome loss and chromosome gain following brief exposures to low concentrations of these chemicals, and these nitriles also induced rapid paralysis. Acrylonitrile-induced chromosome loss only but did not induce paralysis. Fumaronitrile, in contrast with the results reported in yeast, was ineffective in inducing chromosome loss or gain. Virtually all exceptional offspring induced by acetonitrile and propionitrile were recovered in the first sampled eggs, corresponding to treated mature oocytes. Additionally, the time interval between treatment and sampling was shown to be important, suggesting rapid loss or detoxification of the nitriles. Genetic analysis demonstrated that most aneuploids resulted from induced segregation errors during the first division of meiosis. Cold treatments were found to be ineffective in enhancing the effects of acetonitrile, suggesting important differences between the Drosophila and yeast aneuploidy detection systems. Possible mechanisms by which nitriles may disrupt chromosome segregation in Drosophila oocytes are considered.     

Culture expansion induces non-tumorigenic aneuploidy in adipose tissue-derived mesenchymal stromal cells

Background aimsAdipose tissue-derived mesenchymal stromal cells (ASCs) are of interest as a cell therapeutic agent for immunologic and degenerative diseases. During in vitro expansion, ASCs may be at risk for genetic alterations, and genetic screening is a prerequisite. We examined the presence of aneuploidy in ASCs and its origin and development during culture and evaluated the implications of aneuploidy for therapeutic use of ASCs.MethodsAdipose tissue of healthy individuals was used for isolation and expansion of ASCs. Chromosome copy numbers were studied using fluorescence in situ hybridization analysis. Aneuploidy was studied in freshly isolated ASCs, in ASCs cultured for 0–16 passages and in senescent cultures. To evaluate the plasticity of ploidy, ASCs were cloned, and the variation of ploidy in the clones was examined. Tumorigenicity was studied by subcutaneous injection of aneuploid ASCs in immunodeficient NOD/SCID mice.ResultsNo aneuploidy was detected in freshly isolated ASCs. In low passages (passages 0–4), aneuploidy was detected in 3.4% of ASCs. Prolonged culture expansion of ASCs (passages 5–16) resulted in a significant increase of aneuploidy to 7.1%. With senescence, aneuploidy increased further to 19.8%. Aneuploidy was observed in clones of diploid ASCs, demonstrating the de novo development of aneuploidy. No transformation of ASCs was observed, and in contrast to cancer cell lines, aneuploid ASCs were incapable of tumor formation in immunodeficient mice.ConclusionsASC cultures contain a stable percentage of aneuploid cells. Aneuploidy was not a predecessor of transformation or tumor formation. This finding indicates that aneuploidy is culture-induced but unlikely to compromise clinical application of ASCs.     

PVF2, a PDGF/VEGF-like growth factor,induces hemocyte proliferation in Drosophila larvae

Blood cells play a crucial role in both morphogenetic and immunological processes in Drosophila, yet the factors regulating their proliferation remain largely unknown. In order to address this question, we raised antibodies against a tumorous blood cell line and identified an antigenic determinant that marks the surface of prohemocytes and also circulating plasmatocytes in larvae. This antigen was identified as a Drosophila homolog of the mammalian receptor for platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF). The Drosophila receptor controls cell proliferation in vitro. By overexpressing in vivo one of its putative ligands, PVF2, we induced a dramatic increase in circulating hemocytes. These results identify the PDGF/VEGF receptor homolog and one of its ligands as important players in Drosophila hematopoiesis.     

Intracellular protein binding to asbestos induces aneuploidy in human lung fibroblasts

Exposure to the natural mineral fiber asbestos causes severe lung-damaging fibrosis and cancer, yet it continues to be used as an industrial insulating material throughout the world. When cultured human lung cells are exposed to asbestos, individual fibers are engulfed into the cytoplasm where they induce significant mitotic aberrations leading to chromosomal instability and aneuploidy. The mechanisms of how asbestosis ultimately leads to lung cancer remain unclear. However, our experiments indicate that intracellular asbestos fibers induce aneuploidy and chromosome instability by binding to a subset of proteins that include regulators of the cell cycle, cytoskeleton, and mitotic process. Moreover, precoating of fibers with protein complexes efficiently blocked asbestos-induced aneuploidy in human lung cells without affecting their uptake by cells. These results provide new evidence that asbestos fibers can contribute to significant spindle damage and chromosomal instability by binding to proteins needed for the assembly and regulation of the cytoskeleton or the cell cycle.     

The induction of aneuploidy by colcemid fed to Drosophila melanogaster females

Colcemid, a chemical closely related to colchicine, was fed to Drosophila melanogaster females (0.0001 and 0.0005%, respectively). In the F₁ the frequency of aneuploid males (XO karyotype) and aneuploid females (either of the XXY or of the XXXY karyotype, with 2 and 3 sets of autosomes, respectively) was significantly higher than in the controls as shown by genetical methods supplemented by cytological tests. A consistent brood pattern effect was observed, possibly but not necessarily reflecting differential stage sensitivity to the action of colcemid. It seems plausible to assume that most of the aneuploid exceptions were produced via colcemid-induced spindle defects leading to lagging of the X-chromosome. Exclusion of the lagging X from the pronucleus (or its precursors) would yield XO males, its inclusion would yield XXY females. Definitely more XO than XXY exceptions were observed.     

dXNP, a Drosophila homolog of XNP/ATRX, induces apoptosis via Jun-N-terminal kinase activation

XNP/ATRX, a causative gene of X-linked alpha-thalassemia/mental retardation syndrome, encodes an SNF2 family ATPase/helicase protein. To better understand the role of XNP/ATRX in development, we isolated and characterized a Drosophila XNP/ATRX homolog, dXNP, which contains highly conserved SNF2 and helicase domains. Ectopically expressed dXNP induced strong apoptosis in the developing eye and wing, but did not affect cell cycle progression or the expression of wingless and engrailed, essential regulators of development. The dXNP-induced apoptosis was strongly suppressed by DJNKK/hemipterous mutation, and dXNP increased JNK activity. Taken together, these results suggest that dXNP regulates apoptosis via JNK activation.     

Short term cyclin D1 overexpression induces centrosome amplification, mitotic spindle abnormalities, and aneuploidy

In normal cells, cyclin D1 is induced by growth factors and promotes progression through the G(1) phase of the cell cycle. Cyclin D1 is also an oncogene that is thought to act primarily by bypassing the requirement for mitogens during the G(1) phase. Studies of clinical tumors have found that cyclin D1 overexpression is associated with chromosome abnormalities, although a causal effect has not been established in experimental systems. In this study, we found that transient expression of cyclin D1 in normal hepatocytes in vivo triggered dysplastic mitoses, accumulation of supernumerary centrosomes, abnormalities of the mitotic spindle, and marked chromosome changes within several days. This was associated with up-regulation of checkpoint genes p53 and p21 as well as hepatocyte apoptosis in the liver. Transient transfection of cyclin D1 also induced centrosome and mitotic spindle abnormalities in breast epithelial cells, suggesting that this may be a generalized effect. These results indicate that cyclin D1 can induce deregulation of the mitotic apparatus and aneuploidy, effects that could contribute to the role of this oncogene in malignancy.     

Use of a cell hybrid test system to demonstrate that benomyl induces aneuploidy and polyploidy

We have monitored the segregation of a single human chromosome in a human-Chinese hamster hybrid cell line, EUBI, following exposure to benomyl. We found a dose-dependent increase in frequency of aneuploidy, but a much more marked induction of polyploidy was noted at the highest benomyl concentration. We confirm the usefulness of this assay for determining genetic risk associated with human exposure to environmental chemicals.     

Bacterial repopulation of drinking water pipe walls after chlorination

The short-term kinetics of bacterial repopulation were evaluated after chlorination of high-density polyethylene (HDPE) colonized with drinking water biofilms and compared with bare HDPE surfaces. The effect of chlorination was partial as a residual biofilm persisted and was time-limited as repopulation occurred immediately after water resupply. The total number of bacteria reached the same levels on both the bare and chlorinated biofilm-fouled HDPE after a seven-day exposure to drinking water. Due to the presence of a residual biofilm, the hydrophobicity of chlorinated biofilm-fouled surface exhibited much lower adhesion forces (2.1 nN) compared to bare surfaces (8.9 nN). This could explain the rapid repopulation after chlorination, with a twofold faster bacterial accumulation rate on the bare HDPE surface. γ-Proteobacteria dominated the early stages of repopulation of both surfaces and a shift in the dominance occurred over the colonization time. Such observations define a timescale for cleaning frequency in industrial environments and guidelines for a rinsing procedure using drinking water.     

Drinking water chlorination in dairy beef fattening bulls: water quality,potential hazards,apparent total tract digestibility,and growth performance

The first study aimed to evaluate the effect of drinking water disinfection (chlorination: NaClO 15%) and conditioning (acidification: H₃PO₄ diluted 1:5 in water) on water quality, water and feed consumption, apparent total tract digestibility, and its potential hazardous effects on Holstein bulls fed high-concentrate diets. Twenty-four animals (221 ± 20.9 kg of BW, and 184 ± 9.9 days of age) were individually assigned to one of four treatments according to a 2 × 2 factorial arrangement: conditioning (with or without acidification) and disinfection (with or without chlorination). The entire study lasted 210 days. Physicochemical and microbiological water quality, water and feed consumption, haematological and biochemical blood parameters, and apparent total tract digestibility were measured; data were analysed via a mixed-effects model. Chlorination and acidification increased (P = 0.02) free residual chlorine in water, and chlorination reduced (P = 0.01) total coliform and Clostridium perfringens counts in water. Treatment did not affect water consumption, total DM intake, or blood parameters. At the beginning of the study, NDF digestibility decreased (P = 0.04) with acidification, however, this was restored at the end of the study. The second study evaluated the potential benefit of drinking water chlorination and acidification on the performance of crossbred Holstein bulls fed high-concentrate diets under commercial conditions. Ninety-six animals (322 ± 35.0 kg of BW, and 220 ± 14.2 days of age) were allocated into six pens assigned to one of the two treatments: untreated drinking water or drinking water treated with chlorination and acidification for a total of 112 days. Physicochemical and microbiological water quality, water and concentrate consumption, eating behaviour, growth performance, and carcass quality were analysed via a mixed-effects model. Water conditioning and disinfection increased (P = 0.01) free residual chlorine concentration and reduced (P = 0.04) total coliform count in water. Although water consumption and eating behaviour were similar between treatments, water conditioning and disinfection increased average daily weight gain (P = 0.03), BW before slaughter (P = 0.01), and hot carcass weight (P = 0.01). In conclusion, drinking water chlorination and acidification in fattening dairy beef bulls is recommended as it improves growth performance without any detrimental side effects on health or nutrient digestibility.