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. Although glyphosate is commonly considered to be relatively non-toxic, we utilized in vitro DNA microarray analysis of this chemical to evaluate its capacity to alter the expression of a variety of genes in human cells. We selected a group of genes, determined by DNA microarray analysis to be dysregulated, and used quantitative real-time PCR to corroborate their altered states of expression. We discussed the reported function of those genes, with emphasis on altered physiological states that are capable of initiating adverse health effects that might be anticipated if gene expression were significantly altered in either adults or embryos exposed in utero.

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. DNA microarray analysis of diazinon-treated cells showed significant up- or down-regulation of a large number of genes compared to untreated cells. Of the 600 human genes on the Phase 1 chip utilized for these studies, two specific genes--calreticulin and TGF-beta3--were selected for corroboration using quantitative real time PCR (qrtPCR). qrtPCR, completed to assess gene expression levels for calreticulin and TGFbeta3, confirmed results showing significant up-regulation of these two genes obtained from the microarray data. These studies were designed to provide baseline data on the gene expression-altering capacity of a specific chemical, diazinon, and allow a partial assessment of the potentially deleterious effects associated with exposure of human cells to this chemical. Currently, it is not known whether results from cells in vitro can be extrapolated to human health consequences of chemical exposure.

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. DNA microarray analysis showed significant up- or down-regulation of a number of genes in treated cells compared to untreated cells. Of the 600 human genes on the chip utilized, specific genes with related functions were selected for additional consideration. Real time quantitative PCR (qrtPCR) completed to corroborate mRNA levels as a measure of specific gene expression, confirmed results obtained from analysis of the micro-array data. The data show that ERCC5, encoding Xeroderma pigmentosum protein G (XPG), essential for DNA excision repair, and ribonucleotide reductase subunit M1 (RNRM1), encoding a gene necessary for providing the nucleotides needed for DNA repair, were down-regulated in cells treated with diazinon. These studies were designed to provide base-line data on the gene expression-altering capacity of a specific agricultural chemical, diazinon, and allow assessment of some of the potentially deleterious effects associated with exposure of human cells to diazinon.

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. The results suggest that DEHP, a known estrogen agonist and probable androgen antagonist, alters the expression of a number of genes, many of which are critical for fetal development. Down-regulation of two genes, FGD1 and PAFAH1B1, related in that both are essential for fetal brain development, was corroborated using quantitative real time PCR. These studies show DEHP to be a highly effective human gene expression-altering chemical, and that, at appropriate concentrations, it has the possibility of altering fetal central nervous system development, resulting in the birth defects lissencephaly and/or faciodigitogenital dysplasia.

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. DKN1 was the first clone to be evaluated for future research use, and has been continuously cultured for 8 years. Intracellular cytokeratin and the expression of Tag were determined in DKN1, and cell growth was evaluated under different concentrations of l-glutamine, glutathione, and N-acetylcysteine. DKN1 cells did not require high levels of l-glutamine as previously reported for cetacean cells, and addition of antioxidants at the concentrations used in this study (2.0mM) decreased the rate of cell division. These data suggest strongly that these immortalized bottlenose dolphin epithelial cells have different levels of, and requirements for, glutathione than would be considered normal for terrestrial mammalian cells, do not require high levels of l-glutamine as previously suggested for dolphin cells, and exhibit decreased levels of cell growth and viability in high levels of the antioxidant GSH and its precursor, NAC.

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. Cells of an oestrogen-dependent human breast cancer-derived line, MCF-7, were treated with a range, 0.033-3.3 ppb (ng/mL), of Enable, and gene expression was compared to that of untreated cells. Microarray analysis using a chip with 600 gene spots showed downregulation of eight genes and upregulation of 34 genes in cells treated with 3.3 ppb of Enable, compared to untreated cells. Specific genes were selected for consideration. Real-time PCR confirmed results obtained from analysis of the microarray data for the genes phenol sulphotransferase (PST), intercellular adhesion molecule-1 (ICAM-1), transforming growth factor beta-3 (TGF beta-3) and calreticulin. These studies were designed to provide base-line data on the gene expression-altering capacity of a specific chemical at a low dose, and will allow assessment of the possible deleterious effects that may be caused in human cells by exposure to the agricultural chemical Enable.

Dose response studies of acute feline immunodeficiency virus PPR strain infection in cats.

The effects of virus dose on host response were evaluated for the PPR strain of feline immunodeficiency virus (FIV-PPR). Specific pathogen-free cats were inoculated intravenously with 50, 250 or 1250 TCID(50) of FIV-PPR. Two weeks after inoculation, virus was detected in 10(6) peripheral blood mononuclear cells (PBMCs) of all infected animals, and the CD4(+):CD8(+) T lymphocyte ratios fell from greater than 2 to approximately 1 in all infected animals within the first 8 weeks after infection. Provirus detected in all groups using PCR and 10(3) PBMC was biphasic. Nine of 15 animals were positive between weeks 2 and 4 p.i. and 14 of 15 were positive by week 8 p.i. Transient lymphadenopathy was detected in most cats receiving 1250 TCID(50) and the 250 TCID(50) of virus, whereas no lymphadenopathy was detected in the 50 TCID(50) group or the five uninfected cats. Animals that had received the largest dose seroconverted earliest (on average at week 4.0) and those receiving the least seroconverted last (on average at week 5.6). Neither neutropenia nor lymphopenia were detected. FIV-specific CTL responses of memory effector cells could be detected in animals receiving all three doses but was highly variable among individual animals. Neurological manifestations determined after 15 weeks p.i. were observed in most infected cats, including two of the three that had received 50 TCID(50) of virus. However, the observed neurologic abnormalities were markedly less severe in the animals receiving the least amount of virus. Therefore, lymphadenopathy and neurologic signs of illness were less severe and seroconversion was slower in the animals that received the lowest dose compared with those receiving the 250 and 1250 TCID(50) doses of the FIV-PPR strain.

Anti-feline immunodeficiency virus (FIV) soluble factor(s) produced from antigen-stimulated feline CD8(+) T lymphocytes suppresses FIV replication.

Feline immunodeficiency virus (FIV) causes AIDS-like symptoms in infected cats. Concanavalin A (ConA)-stimulated peripheral blood mononuclear cells (PBMC) from chronically FIV strain PPR-infected cats readily expressed FIV. In contrast, when PBMC from these animals were stimulated with irradiated, autologous antigen-presenting cells (APC), at least a 10-fold drop in viral production was observed. In addition to FIV-specific cytotoxic T lymphocytes, anti-FIV activity was demonstrated in the cell-free supernatants of effector T lymphocytes stimulated with APC. The FIV-suppressive activity was induced from APC-stimulated PBMC of either FIV-infected or uninfected cats but not from ConA-stimulated PBMC. Suppression of FIV strain PPR replication was observed for both autologous and heterologous feline PBMC, was dose dependent, and demonstrated cross-reactivity and cell specificity. It was also demonstrated that the anti-FIV activity originated from CD8(+) T lymphocytes and was mediated by a noncytolytic mechanism.

Timing of the response of coleoptiles to the application and withdrawal of various auxins.

A study was made of the time courses of growth promotion and the reversal of growth promotion upon the addition and withdrawal of various auxins. Growth promotion by 1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) occurs more slowly and is less vigorous than growth promotion by the same concentration of indoleacetic acid (IAA).The time required for the reversal of the stimulation of elongation by auxin is many times greater for 2,4-D-stimulated growth than for IAA- or NAA-stimulated growth (80 min vs. about 10 min). This difference appears to be due to the sluggish exit of 2,4-D since (1) experiments with labeled auxins show that 2,4-D moves out of the tissue more slowly than IAA, and (2) it is possible to shorten the time required for a decline in elongation rate after the removal of 2,4-D to 13 min by adding an auxin antagonist (p-chlorophenoxyisobutyric acid).The rapid reversal of the hormonal stimulation of growth is discussed in relation to possible mechanisms of action of auxin.