DJ-1 mutation decreases astroglial release of inflammatory mediators.

Mutations in DJ-1, reactive gliosis and concomitant inflammatory processes are implicated in the pathogenesis and progression of Parkinson's disease (PD). To study the physiological consequences of DJ-1 mutation in the context of neuroinflammatory insult, primary cortical astrocytes were isolated from DJ-1 knockout mice. Astrocytes were exposed to 1µg/mL lipopolysaccharide (LPS) for 24h following 2h pre-exposure to inhibitors of MEK (U0126), JNK (JNK inhibitor II) or p38 (SB203580). Real-time PCR was used to assess the LPS-induced expression of pro-inflammatory mediators cyclooxygenase 2 (COX2), inducible nitric oxide synthetase (NOS2), and tumor necrosis factor α (TNFα). LPS-induced expression of COX2 decreased similarly in DJ-1(+/+) and DJ-1(-/-) astrocytes in response to inhibition of p38, but was unaffected by inhibition of MEK or JNK. No significant alterations in NOS2 expression were observed in any inhibitor-treated cells. The inhibitors did not affect expression of TNFα; however, DJ-1(-/-) astrocytes had consistently lower expression compared to DJ-1(+/+) counterparts. Secretion of TNFα and prostaglandin E2 (PGE2) into the culture medium was significantly decreased in DJ-1(-/-) astrocytes, and inhibition of p38 decreased this secretion in both genotypes. In conclusion, DJ-1(-/-) astrocytes may provide decreased neuroprotection to surrounding neurons due to alterations in pro-inflammatory mediator expression.

Proteomic analysis of diaminochlorotriazine adducts in wistar rat pituitary glands and LbetaT2 rat pituitary cells.

Atrazine (ATRA) is the most commonly applied herbicide in the United States and is frequently detected in drinking water at significant levels. After oral exposure, ATRA metabolism yields diaminochlorotriazine (DACT), an electrophilic molecule that has been shown to form covalent protein adducts. This research was designed to identify ATRA-induced protein adducts formed in the pituitary gland of ATRA-exposed rats and in DACT-exposed LbetaT2 rat pituitary cells. Immunohistochemistry showed diffuse cytoplasmic and nuclear staining in both pituitary sections and LbetaT2 cells indicating the formation of DACT protein adducts. Protein targets from both rat pituitaries and LbetaT2 cell culture were identified following two-dimensional electrophoresis (2DE), immunodetection, and matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. Western blots from both exposed rats and LbetaT2 cells revealed over 30 DACT-modified spots that were not present in control animals. Protein spots were matched to concurrently run 2DE gels stained with Sypro Ruby, excised, and in-gel-digested with trypsin. Mass spectrometry analysis of digest peptides resulted in the identification of 19 spots and 8 unique proteins in the rats and 21 spots and 19 unique proteins in LbetaT2 cells. The identified proteins present in both sample types included proteasome activator complex subunit 1, ubiquitin carboxyl-terminal hydrolase isozyme L1, tropomyosin, ERp57, and RNA-binding proteins. Each of these proteins contains active-site or solvent-exposed cysteine residues, making them viable targets for covalent modification by DACT.

Changes in gene and protein expression in magnetic field-treated human glioma cells.

Because few cancer studies have examined protein profiles and genetic regulation from a single carcinogen exposure, the objective of this study was to determine genetic change via microarray and to evaluate whether that change was a precursor to cellular protein changes. In separate but experimentally identical studies, human glioma SF767 cells were exposed for 3 h to 60-Hz magnetic fields (sham or 1.2 muT). Microarray results suggested that magnetic field treatment resulted in the up-regulation of 5 genes, whereas 25 genes were down-regulated. The mean abundance of 10 identified proteins was altered following 1.2 muT exposure relative to sham (3 increase, 7 decrease). These studies suggest a limited but complicated response in the glioma cells to the magnetic field treatment.

Overexpression of the erbB-2 proto-oncogene in canine osteosarcoma cell lines and tumors.

The status of the erbB-2 (human epidermal growth factor receptor 2/neu) proto-oncogene in canine osteosarcoma (OSA) has not been reported previously. In this study we used real-time reverse transcriptase polymerase chain reaction to evaluate erbB-2 expression in seven canine OSA cell lines and 10 canine OSA tissue samples. We determined erbB-2 to be significantly overexpressed in 86% (six of seven) of the cell lines and 40% (4 of 10) of the OSA tissues samples. Given the importance of erbB-2 in human breast cancer, the finding of erbB-2 overexpression in canine OSA may be important in further understanding the pathogenesis and possible therapies of OSA.

Genetic, biochemical, and characterization of neurological mutant 3, a new mouse model for Parkinson's disease

We have identified a new mutant mouse that we have named new mouse neurological mutant 3 (NM3); it may be a useful model to understand the underlying molecular and genetic basis of Parkinson's disease (PD). A mouse carrying the NM3 mutation arose spontaneously in an RIIIS/J breeding colony and was identified as having a movement disorder. Upon neurological examination of these mice, their movement was found to be slow and abnormal, with characteristic choreaform and bradykinetic-type movements, typical of PD. The importance of the gene mutation in NM3 in the molecular pathway involved in this pathology is underscored by the fact that these mice do not survive past weaning age if they are homozygous for the genetic mutation. We localized the gene mutation by positional cloning and genetic mapping to mouse chromosome 2 in an area that corresponds to human chromosome 2q24-31, which does not contain any known genes associated with PD. However, there was a significant decrease of 15-20 in the levels of dopamine, and its principal metabolite, 3,4-dihydroxyphenylacetic acid, in the midbrain of affected mice. Low concentrations of these substances are associated with PD in human patients, making these mutant mice candidates for studies of this disease

2,3,7,8-Tetrachlorodibenzo-p-dioxin alters hippocampal astroglia-neuronal gap junctional communication.

Halogenated aromatic hydrocarbons (HAHs) such as dibenzo-p-dioxins are known to alter cognitive function. However, the cellular basis of this disruption is not well understood. One possible deleterious effect of exposure to HAHs could be on gap junctional intercellular communication (GJIC) between neurons and astroglia in the brain. As such, this study examined the effects of the highly toxic prototypic HAH, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on GJIC in rat hippocampal primary cell culture. Initial measurements of fluorescence recovery after photobleaching (gap-FRAP) showed dye transfer between astroglia and neurons. N-octanol, a lipophilic alcohol known to uncouple cells by decreasing the open probability of gap junctional channels blocked astroglial-neuronal (A-N) communication as well as astroglial-astroglial (A-A) communication. TCDD initially downregulated GJIC between neurons and astroglia of treatment, but had no effect on astroglial cell pairs. These results indicate the presence of GJIC between neurons and astroglia in culture and demonstrate different sensitivities of gap junction responses to TCDD in homologous and heterologous cell pairs. The finding that 2,3,7,8-TCDD disrupts GJIC through A-N but not A-A channels may have important implications for impaired brain function resulting from developmental exposure to TCDD.

Molecular analysis of gene conversion in spermatids from transgenic mice.

Investigations into the mechanisms and properties of gene conversion in mammals are greatly restricted by the inability to recover all the products of a meiosis. Additionally, the study of this process has been hampered by the lack of visible markers to detect gene conversion, especially when the events are rare. In previous work, we developed a transgenic system for detection and quantitation of gene conversion events in the germline of mice (Murti, J.R., Bumbulis, M., Schimenti, J.C., 1992. High frequency germline gene conversion in transgenic mice. Mol. Cell. Biol. 12, 2545-2552) that could be exploited as an assay for recombinogenic chemicals (Murti, J.R, Schimenti, K.J., Schimenti, J.C., 1994. A recombination-based transgenic mouse system for genotoxicity testing. Mutat. Res. 307, 583-595). A specific intrachromosomal gene conversion event between two complementarily defective lacZ genes resulted in the production of beta-galactosidase in spermatids, enabling a measurement of conversion frequency. Here, we report that the anticancer drug, cisplatin, increased gene conversion in meiotic stage cells in these transgenic mice. Furthermore, a method was developed for direct molecular analysis of transgene conversion events in single or pooled lacZ-positive spermatids. The ability to identify gametes that have undergone a rare gene conversion event, followed by molecular amplification of the recombinant gene, should make it possible to investigate the mechanisms of genetic recombination in mammals in greater detail than previously possible.

Cisplatin increases meiotic crossing-over in mice.

Genetic mapping of traits and mutations in mammals is dependent upon linkage analysis. The resolution achieved by this method is related to the number of offspring that can be scored and position of crossovers near a gene. Higher precision mapping is obtained by expanding the collection of progeny from an appropriate cross, which in turn increases the number of potentially informative recombinants. A more efficient approach would be to increase the frequency of recombination, rather than the number of progeny. The anticancer drug cisplatin, which causes DNA strand breakage and is highly recombinogenic in some model organisms, was tested for its ability to induce germ-line recombination in mice. Males were exposed to cisplatin and mated at various times thereafter to monitor the number of crossovers inherited by offspring. We observed a striking increase on all three chromosomes examined and established a regimen that nearly doubled crossover frequency. The timing of the response indicated that the crossovers were induced at the early pachytene stage of meiosis I. The ability to increase recombination should facilitate genetic mapping and positional cloning in mice.

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure in primary rat astroglia: identification of biochemical and cellular targets.

This paper reports the results from in vitro experiments utilizing vital fluorescent probes and biochemical assays to examine the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) and related compounds in primary rat astroglia in an effort to identify the cellular site(s) involved in toxicity. Application of 100 nM 2,3,7,8-TCDD, a strong Ah receptor agonist, resulted in altered astroglial intracellular Ca2+, a significant decrease in glutathione, a disrupted mitochondrial membrane potential, a significant decrease in glutamine synthetase immunoreactivity and eventual loss of pH maintenance. In contrast, application of 10 microM 1,2,3,4-TCDD, a weak Ah receptor agonist, had no effect on any parameters measured. These findings, coupled with the identification of the 9-10S cytosolic Ah receptor in cultured rat astroglia, are consistent with typical structure-activity relationships observed for other Ah receptor mediated responses. However, the time course of the Ca2+, as well as other responses observed in this study, suggest that the above effects may not necessarily involved the formation of the nuclear Ah receptor complex.

Evidence for cyclophosphamide-induced gene conversion and mutation in mouse germ cells.

Cyclophosphamide (CP) is a widely used antineoplastic drug. It tests positive in several genotoxicity assays, including those with endpoints such as chromosomal aberrations in mammalian cells, mitotic recombination in Drosophila melanogaster, and dominant lethal mutations in rodents. We have explored the effects of CP on genome stability of mouse (Mus domesticus) spermatogenic cells, using a recombination-based transgenic assay called MUSCATEER. In this system, intrachromosomal gene conversion events between two mutually defective lacZ genes generates beta-galactosidase activity in spermatids. The frequency of gene conversion events is determined by scoring spermatids stained with the lacZ substrate, X-gal. A dose-dependent induction of lacZ-positive spermatids was observed following single intraperitoneal CP exposures of 10, 100, and 200 mg/kg. At 200 mg/kg, there was a 25-fold increase over baseline. Treatment of a control transgenic line containing only a frame-shifted lacZ transgene provided an indication that CP also induced reversion mutations. The timing of the response indicated that the induction of recombination and/or mutation occurred primarily in meiotic stage cells. These results demonstrate potent germline mutagenicity of CP, and validate the utility and sensitivity of genetic recombination as a rapid indicator of genotoxicity in whole animals.

Stimulation of calcium uptake in cultured rat hippocampal neurons by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

This study examined the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) and related compounds on the uptake of intracellular calcium ([Ca2+]i) in primary cultures of rat hippocampal neuronal cells. [Ca2+]i levels were detected and quantified by interactive laser cytometry with microscopic image analysis. Cells were noninvasively labeled with fluo-3/AM and all experiments were conducted on cultured rat hippocampal neurons 14 days in culture. Treatment of cell cultures with 2,3,7,8-TCDD (10-100 nM) resulted in a rapid concentration-dependent increase in [Ca2+]i associated with a decrease in mitochondrial membrane potential and activation of alpha-protein kinase C (alpha-PKC). In contrast, 1,2,3,4-TCDD, a weak Ah receptor agonist, had no effect on [Ca2+]i at concentrations as high as 10 microM and similar results were also observed for 2,2',5,5'-tetrachlorobiphenyl. Maximal [Ca2+]i was observed within 30 s after addition of 2,3,7,8-TCDD and remained elevated (at higher concentrations) above resting levels for the duration of the experiment. This rapid increase in [Ca2+]i was blocked by addition of EDTA (2 mM) to the external medium or by pretreatment of the cells with the calcium channel antagonist nifedipine (10 microM). However, pretreatment of the cells with 100 microM cycloheximide failed to block calcium uptake in neuronal cells. These data indicate that rat hippocampal neuronal cells are responsive to 2,3,7,8-TCDD; however, the mechanism is not associated with altered gene transcription and may involve cellular targets.

The need for cellular, biochemical, and mechanistic studies.

The results of diverse in vitro neurotoxicity studies demonstrate that there are variations in cellular responsiveness between different types of neural cells. In contrast to experimental systems that have reported cellular responses to relatively high concentrations of various PCB congeners, our studies with rat hippocampal neural cells indicate that neurons and astroglia are responsive to relatively low levels of TCDD. However, these responses are probably not mediated through the classical Ah receptor pathway, which involves nuclear Ah receptor-mediated modulation of gene expression. It has recently been reported that TCDD-induced phosphorylation and other responses can be observed in some cell lines within minutes after treatment (20), and that cell membrane or cytosolic receptors may also play a role in mediating these effects. Future studies are required to determine both Ah receptor-dependent and -independent pathways associated with the neurotoxicity of PCBs, TCDD, and related compounds. The report that low-level dietary or background exposure to HAHs (32) results in neurobehavioral deficits is still a perplexing problem also requiring additional research and consideration of other dietary factors that may contribute to these effects. For example, we have recently been comparing the toxic effects and relative potencies of TCDD (exodioxins) and other "natural occurring" compounds (endodioxins) such as indole-3-carbinol (vegetables) and polynuclear aromatic hydrocarbons (PAHs, cooked foods), which also bind to the Ah receptor. Dr. Clynn Wilker has shown that in utero exposure of rats to indole-3-carbinol and chrysene (a PAH) cause demasculinization of the adult offspring as previously reported for TCDD (19). Thus, the neurotoxicity of low-level dietary exposure to HAHs should at least consider other possible confounding factors, including dietary endodioxins.

Acute and chronic effects of melatonin as an anticonvulsant in male gerbils.

Melatonin, a hormone produced in the pineal gland and released into the general circulation on a diurnal basis, has been implicated in many behavioral processes, where it has been shown to have anxiolytic, sedative, and anticonvulsant effects. Male gerbils (Meriones unguiculatus) injected daily with melatonin (25 micrograms, s.c.) exhibited a reduced seizure response to pentylenetetrazol (PTZ, 60 mg/kg, s.c.). The present studies determined 1) whether melatonin's effect was related to the time of day that it was administered and 2) whether a single acute injection of melatonin at various doses could produce anticonvulsant activity. Gerbils provided with 13 weeks of daily melatonin injections (25 micrograms, s.c.) exhibited fewer convulsions after PTZ treatment irrespective of the time of day melatonin was injected. In addition, the melatonin-treated gerbils had lower mortality rates (1/12) than the untreated or vehicle-injected gerbils (5/12). On the other hand, single acute injections of melatonin (0.1-10 mg/kg, i.p.) produced no anticonvulsant activity. It appears that the anticonvulsant effects of melatonin occur only after the animals are chronically exposed to the indole. In addition, melatonin's anticonvulsant ability may utilize a different mechanism than those involved in its endocrine effects, since no diurnal difference in melatonin's anticonvulsant activity was observed.

gamma-Aminobutyric acid, catecholamine and indoleamine determinations from the same brain region by high-performance liquid chromatography with electrochemical detection.

A new procedure for the measurement of gamma-aminobutyric acid, norepinephrine, dopamine, serotonin and 5-hydroxyindoleacetic acid from the same brain region was developed. In general, two separate high-performance liquid chromatographic runs were performed, one for the gamma-aminobutyric acid determination and one for the determination of the monoamines. The electrochemical detection of gamma-aminobutyric acid was determined by a new procedure that utilized a small aliquot of the brain sample prepared for monoamine measurement. This assay was linear and parallel between 6 and 200 ng per 20-microliters injection with 5-aminovaleric acid utilized as an internal standard. Inter-assay variability averaged 5% throughout the assay with gamma-aminobutyric acid values in the gerbil hypothalamus of 344 micrograms/g. The catecholamine assay has been characterized previously and utilizes 3,4-dihydroxybenzylamine as an internal standard with less than 5% variability. Norepinephrine, dopamine, serotonin and 5-hydroxyindoleacetic acid levels in the gerbil hypothalamus averaged 2922, 729, 797 and 272 ng/g, respectively. This new protocol allows a wide range of neurochemicals to be determined and evaluated from the same brain region.