Alteration of estrogen-regulated gene expression in human cells induced by the agricultural and horticultural herbicide glyphosate.

Gene expression is altered in mammalian cells (MCF-7 cells), by exposure to a variety of chemicals that mimic steroid hormones or interact with endocrine receptors or their co-factors. Among those populations chronically exposed to these endocrine disruptive chemicals are persons, and their families, who are employed in agriculture or horticulture, or who use agricultural/horticultural chemicals. Among the chemicals most commonly used, both commercially and in the home, is the herbicide glyphosate.

Comparison of reactive oxygen scavenging systems between a cetacean (DKN1) and a porcine renal epithelial cell line (LLC-PK1).

A transformed renal epithelial cell line, (DKN(1)), from an Atlantic Bottlenose Dolphin, Tursiops truncatus was established in this laboratory and has been used for in vitro genomic analysis and initial toxicological evaluations of dolphin cells. Studies were initiated to compare maintenance of normal antioxidant mechanisms in DKN(1) with similar mechanisms in cells of a pig kidney line, LLC-PK(1). Levels of catalase, glutathione peroxidase, and of reduced glutathione in these dolphin cells were significantly lower than in the porcine cells.

DEHP, bis(2)-ethylhexyl phthalate, alters gene expression in human cells: possible correlation with initiation of fetal developmental abnormalities.

Diethylhexylphthalate (DEHP) is a widely distributed phthalate, to which humans are exposed to due to its variety of commercial and manufacturing uses. As a plasticiser, it is found in a wide number of products, and metabolites of DEHP have been detected in urine samples from a high percentage of the people screened for phthalates. We utilised DNA microarray analysis to evaluate DEHP for gene expression disrupting activity using the human cell line MCF-7, and found that DEHP significantly dysregulated approximately 34% of the 2400 genes spotted on the NEN2400 chip we used.

Alteration of gene expression in human cells treated with the agricultural chemical diazinon: possible interaction in fetal development.

Agricultural chemicals frequently alter human health or development, typically because they have endocrine agonist or antagonist activities and alter hormone-regulation of gene expression. The insecticide, diazinon, was evaluated for gene expression disrupting activity using MCF-7 cells, an estrogen-dependent human cell line, to examine the capacity of the insecticide to disrupt gene expression essential for morphological development, immune system development or function, and/or central nervous system development and function. MCF-7 cells were treated with 30, 50 or 67 ppm diazinon, and gene expression was measured in treated cells compared to expression in untreated or estrogen-treated cells.

Altered gene expression in human cells treated with the insecticide diazinon: correlation with decreased DNA excision repair capacity.

Many industrial and agricultural chemicals have steroid hormone agonist or antagonist activities and disrupt hormone-regulated gene expression. The widely-used agricultural insecticide, diazinon, was evaluated using MCF-7 cells - a breast cancer-derived, estrogen-dependent, human cell line - to examine the capacity of this chemical to alter steroid hormone-regulated gene expression. MCF-7 cells were treated with 30, 50, or 67 ppm of diazinon, and gene expression in treated cells was measured as mRNA levels in the cells compared to mRNA levels in untreated or estrogen-treated cells.

Altered gene expression in human cells induced by the agricultural chemical Enable.

Steroid hormones bind to highly specific nuclear receptors, regulating gene expression that results in normal fetal growth and development and/or in normal adult physiological function. Many industrial and agricultural chemicals may bind one or more nuclear receptors, acting as mimics of steroid hormones, and are called endocrine disruptive chemicals (EDC) because they alter the expression of endocrine-regulated genes. A widely used fungicide, Enable (fenbuconazole), was evaluated to examine its capacity to alter endocrine-regulated gene expression.

Development of an in vitro model of excess intracellular reactive oxygen species.

These investigations characterize an in vitro model for generating excess intracellular reactive oxygen species (ROS). This novel model may be useful in the identification and delineation of cellular mechanisms associated with aging due to the link between age and excess oxidative events. The human cell line, MCF7, was stably transfected using the pSV3.

Disruption of estrogen-regulated gene expression by dioxin: downregulation of a gene associated with the onset of non-insulin-dependent diabetes mellitus (type 2 diabetes).

Expression of an estrogen-regulated reporter gene, growth of MCF-7 cells in the presence of 17beta-estradiol (E2) or E2 plus TCDD, and DNA microarray plus real time quantitative PCR analyses of gene expression in MCF-7 cells were used to evaluate the effects of TCDD, a known E2 antagonist, on E2-regulated gene expression in human cells. TCDD added simultaneously with E2 exhibited significantly decreased E2-associated upregulation of reporter gene expression compared with cells treated with E2 alone, and decreased E2 enhancement of mitosis in MCF-7 cells. MCF-7 cells treated with E2 or E2 plus TCDD and DNA microarray-evaluated to determine patterns of gene expression, showed substantial differences in gene expression in TCDD-treated cells compared with E2-treated cells.

Generation and partial characterization of a transformed cetacean cell line.

A primary epithelial cell line, DK1, established from renal tissue of a spontaneously aborted female Atlantic bottlenose dolphin was transfected with linearized pSV3.neo, an SV40 virus-derived plasmid encoding large tumor antigen (Tag). Transfected cells were grown in cetacean culture medium supplemented with 400 microg/ml geneticin (G418), and individual clones were selected using cloning rings.

Replicative enzymes, DNA polymerase alpha (pol alpha), and in vitro ageing.

Normal cells in culture are used to investigate the underlying mechanisms of DNA synthesis because they retain regulatory characteristics of the in vivo replication machinery. During the last few years new studies have identified a number of genetic changes that occur during in vitro ageing, providing insight into the progressive decline in biological function that occurs during ageing. Maintaining genomic integrity in eukaryotic organisms requires precisely coordinated replication of the genome during mitosis, which is the most fundamental aspect of living cells.

Replicative enzymes and ageing: importance of DNA polymerase alpha function to the events of cellular ageing.

A hallmark of cellular ageing is the failure of senescing cells to initiate DNA synthesis and transition from G1 into S phase of the cell cycle. This transition is normally dependent on or concomitant with expression of a set of genes specifying cellular proteins, some of which directly participate in DNA replication. Deregulation of this gene expression may play a pivotal role in the ageing process.

Activity of DNA polymerase alpha in aging human fibroblasts.

During the past decade intense investigation has focused on cellular aging with the expectation of discovering factors that regulate the replication complex and contribute to the onset and progression of cellular aging. The most striking feature of cellular aging is the failure of sensing diploid cells to enter or complete S phase of the cell cycle. The G1/S phase transition is an initial critical step in the regulation of proliferation in eukaryotic cells, and significant advances have been made toward understanding the basic mechanisms of aging by identifying components of the macromolecular assemblies participating in the G1/S transition.

Aging and DNA polymerase alpha: modulation by dietary restriction.

Aging is an inevitable characteristic of biological processes in living organisms. For the last several years, investigators have proposed numerous mechanisms to explain the basic understanding of aging and its intervention and have provided many insights into the molecular bases and the biological events that contribute to the progressive decline in function observed during cellular aging. It is probable that a number of interacting factors, such as increased somatic mutations, changes in genetic expression, and decreased efficiency of protein synthesis, may contribute to the age-dependent deterioration of physiological processes.

A rapid and sensitive reporter gene that uses green fluorescent protein expression to detect chemicals with estrogenic activity.

A reporter gene sequence was constructed within a eukaryotic expression vector. The altered plasmid contained 2 sequential estrogen response elements (ERE) coupled to a human phosphoglycerate kinase (PGK) promoter inserted upstream from a cDNA sequence encoding enhanced green fluorescent protein (GFP) with a 3'-polyadenylation signal. The plasmid was linearized and transfected into MCF-7 cells, a human breast cancer-derived line that expresses the estrogen receptor (ER).

Protection from glutathione depletion by a glyconutritional mixture of saccharides.

A complex glyconutritional (GN) mixture of mono-, di-and polysaccharides was investigated to assess its capacity to protect two different types of rodent cells, rat hepatocytes and mouse splenocytes, from depletion of glutathione by a sulfhydryl-reactive mycotoxin, patulin, or by coxsackievirus B3 (CVB3) infection, respectively. Rat hepatocytes were treated with the GN mixture in vitro or received carrier medium only prior to treatment with patulin. When treated with the GN mixture prior to patulin exposure hepatocytes demonstrated protection against depletion of intracellular reduced glutathione (GSH).

A glyconutritional mixture (Ambrotose®) provides some amelioration to mice with coxsackievirus-induced pancreatitis.

Challenge of adolescent male CD-1 mice with a coxsackievirus B3 (CVB3) strain (CVB3m) induces mild to severe destruction of pancreatic acinar cells, but causes no deaths and does not induce hyperglycemia. A weekly parenteral (intraperitoneal) administration of a glyconutritional mixture (Ambrotose® to virus-challenged mice was assessed to determine if there were any benefits to recovery over an eight month period. Virus-challenged mice showed a significant weight loss over the initial five weeks of the experiment, but injection of Ambrotose® to similar virus-challenged mice restored the total body weight to levels found in normal mice.

Homologies between human and dolphin chromosomes detected by heterologous chromosome painting.

Human chromosome-specific probes for the entire karyotype were hybridized to metaphase spreads of the Atlantic bottlenose dolphin, Tursiops truncatus, to directly compare the evolutionary conservation of chromosomal segments between these two distantly related species. All human chromosomal paints, except the Y probe, hybridized to Tursiops counterparts, and every dolphin chromosome was painted except for the smallest submetacentric pair. In our analysis, 36 segments of conserved synteny common to the human and dolphin genomes were identified.

An accessory protein of DNA polymerase alpha declines in function with increasing age.

Isoforms of DNA polymerase alpha (pol alpha/primase; pol alpha) were isolated from the livers of C57BL/6 mice either 3 months old (young) or 13 months old (mature). The 13-month-old mice were from two groups, one in which food was available ad libitum (AL), and one in which calories had been restricted to 60% of the AL intake (CR). The polymerases from young vs.

A DNA polymerase alpha accessory protein exhibits structural and functional similarities to SV40 large tumor antigen.

Untransformed cells have been proposed to require a protein homologous to SV40 large tumor antigen (TAg) which functions as a component of the replicase complex during the initiation of DNA synthesis. By definition, this should be a phosphoprotein which interacts with the retinoblastoma protein (pRb) in G0 or early G1, and is capable of binding to and potentiating the activity of DNA polymerase alpha (pol alpha). This protein should also be an ATP-dependent helicase which interacts with the single-stranded DNA (ssDNA) binding protein, RP-A.

A high resolution GBG-banded karyotype of the Atlantic bottlenose dolphin, Tursiops truncatus: generation of an ideogram, and NOR localization by fluorescence in situ hybridization.

A high resolution karyotype was prepared using GBG-banded chromosomes from kidney epithelial cell lines of the Atlantic bottlenose dolphin, Tursiops truncatus. The karyotype was used to generate a banded ideogram of T. truncatus that will facilitate cytogenetic analyses essential for comparison of dolphin chromosomes with those of potentially related terrestrial vertebrates.

Differential expression of DNA polymerase alpha in normal and transformed human fibroblasts.

The expression of DNA polymerase alpha (pol alpha) was studied in human fibroblast lines W138 (fetal lung) and GM3529 (skin, established from a 66 yr old donor), and their Simian virus 40 (SV40) large tumor antigen (TAg)-transformed corollaries, 2RA and 2-1 respectively. Both SV40-transformed and pSV3.neo (SV40-derived plasmid)-transformed cells express TAg, a virally encoded protein not expressed by the normal parent cell lines.