Rubratoxin B elicits antioxidative and DNA repair responses in mouse brain.

Rubratoxin B (RB) is a mycotoxin with potential neurotoxic effects that have not yet been characterized. Based on existing evidence that RB interferes with mitochondrial electron transport to produce oxidative stress in peripheral tissues, we hypothesized that RB would produce oxidative damage to macromolecules in specific brain regions. Parameters of oxidative DNA damage and repair, lipid peroxidation, and superoxide dismutase (SOD) activity were measured across six mouse brain regions 24 h after administration of a single dose of RB. Lipid peroxidation and oxidative DNA damage were either unchanged or decreased in all brain regions in RB-treated mice compared with vehicle-treated mice. Concomitant with these decreased indices of oxidative macromolecular damage, SOD activity was significantly increased in all brain regions. Oxyguanosine glycosylase activity (OGG1), a key enzyme in the repair of oxidized DNA, was significantly increased in three brain regions--cerebellum (CB), caudate/putamen (CP), and cortex (CX)--but not in the hippocampus (HP), midbrain (MB), and pons/medulla (PM). The RB-enhanced OGG1 catalytic activity in these brain regions was not due to increased OGG1 protein expression, but was a result of enhanced catalytic activity of the enzyme. In conclusion, specific brain regions responded to an acute dose of RB by significantly altering SOD and OGG1 activities to maintain the degree of oxidative DNA damage equal to, or less than, that of normal steady-state levels.

Comparison of base-excision repair capacity in proliferating and differentiated PC 12 cells following acute challenge with dieldrin.

Dieldrin, an organochlorine pesticide and known neurotoxicant, is ubiquitously distributed in the environment. Dieldrin depletes brain monoamines in some animal species and is toxic for dopaminergic neurons in vitro. Dieldrin interferes with mitochondrial electron transport and increases generation of superoxide anion. Reactive oxygen species have been shown to produce oxidative lesions to DNA bases, i.e., 8-hydroxy-2'-deoxyguanosine (8-oxodGuo). Accumulation of 8-oxodGuo has been shown to be promutagenic in proliferating cells, and can lead to degeneration in fully differentiated cells. The objective of this study was to determine the effects of dieldrin exposure on the activity of the enzyme responsible for removing 8-oxodGuo, OGG1, from undifferentiated (untreated with NGF) and differentiated (NGF-treated) PC 12 cells. Proliferating PC 12 cells exhibited a mild upregulation of glycosylase activity, reaching a maximum by 1 h and returning to baseline by 6 h. Differentiated (+) NGF cells showed a time-dependent decline in activity reaching a nadir at 3 h with a return towards baseline by 6 h. Levels of the damaged base, 8-oxodGuo, in the differentiated PC12 cells appeared to be regulated by the activity of OGG1. In contrast, levels of the damaged base in actively proliferating cells were independent of the OGG1 activity. This difference between actively dividing and differentiated cells in the regulation of base-excision repair and DNA damage accumulation explains, in part, the vulnerability of postmitotic neurons to oxidative stresses and neurotoxins.

Expression of neural markers in human umbilical cord blood.

A population of cells derived from human and rodent bone marrow has been shown by several groups of investigators to give rise to glia and neuron-like cells. Here we show that human umbilical cord blood cells treated with retinoic acid (RA) and nerve growth factor (NGF) exhibited a change in phenotype and expressed molecular markers usually associated with neurons and glia. Musashi-1 and beta-tubulin III, proteins found in early neuronal development, were expressed in the induced cord blood cells. Other molecules associated with neurons in the literature, such as glypican 4 and pleiotrophin mRNA, were detected using DNA microarray analysis and confirmed independently with reverse transcriptase polymerase chain reaction (RT-PCR). Glial fibrillary acidic protein (GFAP) and its mRNA were also detected in both the induced and untreated cord blood cells. Umbilical cord blood appears to be more versatile than previously known and may have therapeutic potential for neuronal replacement or gene delivery in neurodegenerative diseases, trauma, and genetic disorders.

Organ-specific differences in 8-oxoguanosine glycosylase (OGG1) repair following acute treatment with benzo[a]pyrene.

The lung has been shown to be a target organ for the deleterious effects of Benzo[a]pyrene (B[a]P), regardless of the route of exposure. 8-hydroxy-2'-deoxyguanosine (oxo8dG) is a mutagenic lesion formed in DNA following exposure to B[a]P. The objective of this study was to determine the capacity of different organs to repair oxo8dG following intraperitoneal (i.p.) treatment with B[a]P. Male Spraque-Dawley rats were administered 20 mg/kg B[a]P i.p., 2 times/day for 5 days. A 26% decrease in the capacity to remove oxo8dG was observed in lung tissue at 72 hours and recovered 20% above control values at 120 hours. The capacity of the liver and kidney remained at baseline for all time points analyzed. A 7-fold increase in oxo8dG was observed in the lung at 72 hours. This study demonstrates that organ-specific differences exist in the capacity to remove oxo8dG and further demonstrates the susceptibility of lung tissue to the effects of B[a]P.

Alpha1-adrenergic receptors and their significance to chemical-induced nephrotoxicity--a brief review.

Stimulation of alpha-adrenergic receptors by cold stress or adrenergic agents has been shown to potentiate the toxicity of numerous toxicants. Several lines of evidence indicate that this interaction is dependent on glutathione depression and increased cytosolic Ca2+ concentrations produced by alpha1-adrenergic compounds. In this report, evidence is provided in support of the mechanism of adrenoreceptor-mediated potentiation of nephrotoxicity. Alpha1-adrenergic agonists are shown to potentiate the toxicity of nephrotoxicants that exert their toxic effects via glutathione conjugation or Ca2+ deregulation. This review summarizes the effects of the alpha1-adrenergic agonist, phenylephrine, at enhancing the toxicity of 2-bromohydroquinone, 1,2-dibromoethane, and cis-diammineplatinum(II) dichloride.

Adult bone marrow stromal cells differentiate into neural cells in vitro.

Bone marrow stromal cells (BMSC) normally give rise to bone, cartilage, and mesenchymal cells. Recently, bone marrow cells have been shown to have the capacity to differentiate into myocytes, hepatocytes, and glial cells. We now demonstrate that human and mouse BMSC can be induced to differentiate into neural cells under experimental cell culture conditions. BMSC cultured in the presence of EGF or BDNF expressed the protein and mRNA for nestin, a marker of neural precursors. These cultures also expressed glial fibrillary acidic protein (GFAP) and neuron-specific nuclear protein (NeuN). When labeled human or mouse BMSC were cultured with rat fetal mesencephalic or striatal cells, a small proportion of BMSC-derived cells differentiated into neuron-like cells expressing NeuN and glial cells expressing GFAP.

DNA damage, repair, and antioxidant systems in brain regions: a correlative study.

8-Hydroxy-2'-deoxyguanosine (oxo(8)dG) has been used as a marker of free radical damage to DNA and has been shown to accumulate during aging. Oxidative stress affects some brain regions more than others as demonstrated by regional differences in steady state oxo(8)dG levels in mouse brain. In our study, we have shown that regions such as the midbrain, caudate putamen, and hippocampus show high levels of oxo(8)dG in total DNA, although regions such as the cerebellum, cortex, and pons and medulla have lower levels. These regional differences in basal levels of DNA damage inversely correlate with the regional capacity to remove oxo(8)dG from DNA. Additionally, the activities of antioxidant enzymes (Cu/Zn superoxide dismutase, mitochondrial superoxide dismutase, and glutathione peroxidase) and the levels of the endogenous antioxidant glutathione are not predictors of the degree of free radical induced damage to DNA in different brain regions. Although each brain region has significant differences in antioxidant defenses, the capacity to excise the oxidized base from DNA seems to be the major determinant of the steady state levels of oxo(8)dG in each brain region.

The X-gal caution in neural transplantation studies.

Cell transplantation into host brain requires a reliable cell marker to trace lineage and location of grafted cells in host tissue. The lacZ gene encodes the bacterial (E. coli) enzyme beta-galactosidase (beta-gal) and is commonly visualized as a blue intracellular precipitate following its incubation with a substrate, "X gal," in an oxidation reaction. LacZ is the "reporter gene" most commonly employed to follow gene expression in neural tissue or to track the fate of transplanted exogenous cells. If the reaction is not performed carefully-with adequate optimization and individualization of various parameters (e.g.. pH, concentration of reagents, addition of chelators, composition of fixatives) and the establishment of various controls--then misleading nonspecific background X-gal positivity can result, leading to the misidentification of cells. Some of this background results from endogenous nonbacterial beta-gal activity in discrete populations of neurons in the mammalian brain; some results from an excessive oxidation reaction. Surprisingly, few articles have empha sized how to recognize and to eliminate these potential confounding artifacts in order to maximize the utility and credibility of this histochemical technique as a cell marker. We briefly review the phenomenon in general, discuss a specific case that illustrates how an insufficiently scrutinized X-gal positivity can be a pitfall in cell transplantation studies, and then provide recommendations for optimizing the specificity and reliability of this histochemical reaction for discerning E. coli beta-gal activity.

Oxidative DNA damage in the aging mouse brain.

The brain exhibits regional vulnerabilities to many insults, and age itself has differential effects on neuronal populations as exemplified by the age-dependent loss of dopaminergic neurons in the nigrostriatal system. We hypothesized that oxidative damage to DNA was more likely to occur in the nigrostriatal system which undergoes significant neurochemical and functional changes with age. To test this hypothesis, oxidative damage to DNA, indicated by levels of 8-hydroxy2'-deoxyguanosine (oxo8dG), was measured in pons-medulla (PM), midbrain (MB), caudate-putamen (CP), hippocampus (HP), cerebellum (CB), and cerebral cortex (CX) at 3, 18, and 34 months of age in C57/b1 mice. Steady-state levels of oxo8dG increased significantly with age in MB, CP, and CB, but not in PM, HP, or CX. Manganese superoxide dismutase (MnSOD) activity decreased with age in MB, CP, and HP, but not in PM, CB, or CX. Regional activities of Cu/Zn superoxide dismutase (Cu/Zn SOD) and glutathione peroxidase (Glut Px) did not change significantly with age. Concomitant with the regional alterations in DNA damage, there was a significant age-dependent decline in locomotor activity, motor coordination, and striatal dopamine content especially during the interval between 18 and 34 months. In conclusion, oxyradical-associated damage to DNA did not accumulate uniformly across brain regions with age and was highest in brain regions that subserve spontaneous locomotor activity and motor coordination.

Attenuation of age-dependent oxidative damage to DNA and protein in brainstem of Tg Cu/Zn SOD mice.

Age-dependent accumulation of oxidative DNA and protein damage in brainstem and striatum was assessed in normal and transgenic (tg) mice which overexpress human Cu/Zn superoxide dismutase (h-SOD1). A marker of oxidative DNA damage, 8-hydroxy-2'-deoxyguanosine (oxo8dG), was measured at 3, 12, and 18 months of age in control and tg mice. Cu/Zn SOD, but not MnSOD, activities in brainstems and striata from tg mice were increased compared to controls at all ages. At 18 months, oxo8dG levels were increased by 58% in brainstem and by 21% in striatum of control mice. In the tg mice, brainstem and striatal oxo8dG levels were increased to a lesser extent than in the corresponding controls. Protein oxidation (carbonyl content), was increased by 59% at 18 months in control brainstem, but not in striatum, and the increase was significantly attenuated in the tg mice. In summary, oxidative damage to DNA and protein increased with age in brainstem (and to a lesser extent in striatum), and augmented Cu/Zn SOD activity modified the extent of DNA and protein damage.