Generation of reactive oxygen species by xanthine derivatives in MDA-MB-231 human breast cancer cells.

Theophylline reduces cell number in MDA-MB-231 cells through mechanisms over and above phosphodiesterase inhibition. In the current study, we used an intracellular fluorescent dye to show that theophylline and, to a much greater extent, 3-isobutyl-1-methylxanthine, evoke the generation of reactive oxygen species and also sensitize the cells to insult by other oxidants. Xanthine derivatives may therefore offer novel strategies for antitumor therapeutics.

Lead exposure in pheochromocytoma (PC12) cells alters neural differentiation and Sp1 DNA-binding.

Previous studies have revealed that lead modulates the DNA-binding profile of the transcription factor Sp1 both in vivo and in vitro (Dev Brain Res 1998;107:291). Sp1 is a zinc finger protein, that is selectively up-regulated in certain developing cell types and plays a regulatory role during development and differentiation (Mol Cell Biol 1991;11:2189). In NGF-stimulated PC12 cells, Sp1 DNA-binding activity was induced within 48 h of exposure of NGF naïve cells. Exposure of undifferentiated PC12 cells to lead alone (0.1 microM) also produced a similar increase in Sp1 DNA-binding. Since lead altered the DNA-binding profile of Sp1 in newly differentiating cells, neurite outgrowth was assessed as a morphological marker of differentiation to determine whether or not the effects of lead on differentiation were restricted to the initiation phase (unprimed) or the elaboration phase of this process (NGF-primed). NGF-primed and unprimed PC12 cells were prepared for bioassay following exposure to various concentrations of NGF and/or lead. Neurite outgrowth was measured at 48 and 72 h during early stages of NGF-induced differentiation and at 14 h in NGF primed/replated cells. In the absence of NGF, exposure to lead alone (0.025, 0.05, 0.1 microM) promoted measurable neurite outgrowth in unprimed PC12 cells at 48 and 72 h. A similar phenomenon was also observed in primed/replated PC12 cells at 14 h. However, this effect was two to five times greater than unprimed control cells. In the presence of NGF, a similar trend was apparent at lower concentrations, although the magnitude and temporal nature was different from lead alone. In most cases, the administration of higher lead concentrations (1 and 10 microM), in both the absence or presence of NGF, was less effective than the lower concentrations in potentiating neurite outgrowth. These results suggest that lead alone at low doses may initiate premature stimulation of morphological differentiation that may be related to lead-induced alterations in Sp1 binding to DNA.

Does the developmental neurotoxicity of chlorpyrifos involve glial targets? Macromolecule synthesis, adenylyl cyclase signaling, nuclear transcription factors, and formation of reactive oxygen in C6 glioma cells.

The widespread use of chlorpyrifos (CPF) has raised major concerns about its potential to cause fetal or neonatal neurobehavioral damage, even at doses that do not evoke acute toxicity. CPF has been shown to inhibit replication of brain cells, to elicit alterations in neurotrophic signaling governing cell differentiation and apoptosis, and to evoke oxidative stress. However, the specific cell types targeted by CPF have not been clarified, an issue of vital importance in establishing the boundaries of the critical period in which the developing brain is vulnerable. In the current study, we evaluated the effects of CPF on C6 glioma cells, a well-established glial model. In undifferentiated C6 cells, CPF inhibited DNA synthesis in a concentration-dependent manner, with greater potency than had been seen previously with neuronal cell lines. Just as found after in vivo CPF treatment or with neuronal cell lines, the effects on cell replication were independent of cholinergic stimulation, as cholinergic antagonists did not block CPF-induced inhibition. CPF interfered with cell signaling mediated through adenylyl cyclase at the level of G-protein function; the effects again were greater in undifferentiated C6 cells but were still detectable in differentiating cells. In contrast, differentiation enhanced the ability of CPF to elicit the formation of reactive oxygen species and to evoke deficits in Sp1, a nuclear transcription factor essential for differentiation. These results indicate that glial-type cells are targeted by CPF through the same multiple mechanisms that have been demonstrated for the effects of CPF on brain development in vivo. Because glial development continues long after the conclusion of neurogenesis, and given that CPF targets events in both glial cell replication and the later stages of differentiation, the vulnerable period for developmental neurotoxicity of CPF is likely to extend well into childhood.

Is oxidative stress involved in the developmental neurotoxicity of chlorpyrifos?

The increasing use of chlorpyrifos (CPF) has elicited concern about neurotoxic effects on the fetus and neonate. CPF targets a number of events specific to brain development, over and above the ability of its active metabolite, CPF oxon, to inhibit cholinesterase. We used PC12 cells, a model system which displays many of the neurodevelopmental effects of CPF, in order to examine whether oxidative stress underlies the direct effects of CPF on development. Production of reactive oxygen species (ROS) was measured with a fluorescent intracellular dye. When PC12 cell suspensions were treated acutely with CPF for 10 min, ROS generation was increased in a concentration-dependent manner; CPF oxon was much less effective than the native compound. CPF also increased the ROS production in response to an acute sodium nitroprusside challenge, indicating sensitization of the cells to other oxidant stressors. Next, PC12 cells were grown in an undifferentiated state in the presence of CPF or CPF oxon for extended time periods, under conditions in which CPF inhibits mitosis, and the cells were then washed and ROS production measured. Neither compound elicited a significant change in ROS production. Finally, differentiation was initiated with nerve growth factor and the cells were exposed continuously to CPF or CPF oxon over a 72 h period; under these conditions, CPF inhibits neurite outgrowth. When the cells were washed and evaluated for ROS production, no significant differences were seen. These results indicate that CPF, but not CPF oxon, has the ability to elicit acute increases in ROS production. However, the effect disappears immediately once CPF exposure is terminated, possibly reflecting cellular defense mechanisms that lessen the impact of oxidant injury.

Developmental neurotoxicity of chlorpyrifos in vivo and in vitro: effects on nuclear transcription factors involved in cell replication and differentiation.

Chlorpyrifos is a widely used organophosphate insecticide that is a suspected developmental neurotoxin. Although chlorpyrifos exerts some effects through cholinesterase inhibition, recent studies suggest additional, direct actions on developing cells. We assessed the effects of chlorpyrifos on nuclear transcription factors involved in cell replication and differentiation using in vitro and in vivo models. HeLa nuclear protein extracts were incubated with the labeled consensus oligonucleotides for AP-1 and Sp1 transcription factors in the presence and absence of chlorpyrifos. In concentrations previously shown to affect cell development, chlorpyrifos reduced AP-1, but not Sp1 DNA-binding activity. Next, chlorpyrifos was incubated with PC12 cells either during cell replication or after initiation of differentiation with NGF. Chlorpyrifos evoked stage-specific interference with the expression of the transcription factors: Sp1 was reduced in replicating and differentiating cells, whereas AP-1 was affected only during differentiation. Finally, neonatal rats were given apparently subtoxic doses of chlorpyrifos either on postnatal days 1-4 or 11-14 and the effects were evaluated in the forebrain (an early-developing, cholinergic target region) and cerebellum (late-developing region, poor in cholinergic innervation). Again, chlorpyrifos evoked stage-specific changes in transcription factor expression and binding activity, with greater effects on Sp1 during active neurogenesis, and effects on AP-1 during differentiation. The changes were present in both forebrain and cerebellum and were gender-specific. These results indicate that chlorpyrifos interferes with brain development, in part by multiple alterations in the activity of transcription factors involved in the basic machinery of cell replication and differentiation. Noncholinergic actions of chlorpyrifos that are unique to brain development reinforce the need to examine endpoints other than cholinesterase inhibition.

Disruption of the zinc finger domain: a common target that underlies many of the effects of lead.

The health risks associated with exposure to heavy metals such as lead (Pb) remain a major public health concern. The zinc finger is a major structural motif involved in protein-nucleic acid interactions and is present in the largest superfamily of transcription factors. Zinc (Zn) ions coordinate this finger-like structure through bonds created with cysteine and histidine residues. Little information exists on the effects of heavy metals on proteins that contain structural repeats of this kind. Studies by us in the nervous system have shown that factors containing such motifs could be potential targets for perturbation by Pb. We have observed that metals such as Pb interfered with the DNA-binding properties of Sp1 and Egr-1, both in vivo and in vitro. Pb could also directly interfere with the DNA-binding of a recombinant human Sp1 protein. More recently, the effects of Pb on the DNA-binding of the zinc finger protein transcription factor IIIA (TFIIIA) have been demonstrated. Analysis on the effects of Pb on Sp1 revealed that alterations in its DNA-binding were commensurate with changes in the expression of its target genes. The action of Pb on Sp1, Egr-1, and TFIIIA suggests that it can also target other cellular proteins that contain the zinc finger motif and reveals this protein domain as a potential mediator for Pb-induced alterations in protein function. Thus by specifically targeting zinc finger proteins (ZFP), Pb is able to produce multiple responses through its action on a common site that is present in enzymes, channels and receptors.

Sp1 as a target site for metal-induced perturbations of transcriptional regulation of developmental brain gene expression.

Differential gene expression is partially regulated by zinc finger proteins (ZFP) such as Sp1, which may be potential targets for perturbations by environmental metals. In this paper, we discuss the selective effects of lead (Pb) and other heavy metals on the in vitro and in vivo DNA-binding of Sp1, and the developmental expression of its target genes. We have found that the presence of Pb, Zn and Cd in a DNA-binding assay differentially modulated the binding of Sp1 to its specific DNA sequence, while Ca, Mg and Ba, did not. In PC12 cells, cultured in the presence of low concentrations of Pb, a premature enhancement of Sp1 DNA-binding was observed. Similarly, Sp1 DNA-binding in the cerebellum of Pb-exposed animals was shifted to the first week after birth, while the developmental profile of a non-ZFP, NFkB, was not. Furthermore, selective premature peaks of myelin basic protein and proteolipid protein mRNA expression were observed to occur in a manner relative to the changes in Sp1 DNA-binding. Since these genes are high targets for Sp1, these data suggest that exposure to heavy metals may alter developmental gene expression and brain development through selective modulation of the transcriptional activity of Sp1.

Induced hemichrome formation of methemoglobins A, S and F by fatty acids, alkyl ureas and urea.

1. Spectral analysis of the Soret region (450-350 nm) has shown that saturated fatty acids, alkyl ureas and urea induce the conversion of methemoglobins A, S, and F to the hemichrome state. 2. In the presence of fatty acids (C8-C16), methemoglobin F is converted to the hemichrome state more readily than either methemoglobins A or S. 3. Using several alkyl ureas (methyl, ethyl, propyl, butyl), the extent of hemichrome formation was as follows: met Hb F > met Hb S > met Hb A. The ability of these compounds to induce hemichrome formation is related to their increasing hydrophobicity. 4. Conversion to the hemichrome state in the presence of urea (5M) led to the formation of molecular aggregates of hemoglobins S, F and A which may be initiated by subunit dissociation and conformational changes, coupled to increased globin-globin interactions. 5. Similar aggregation occurred for methemoglobin S in the presence of octanoic acid; no significant aggregation was evident for methemoglobin A after 10 hr of exposure to octanoic acid.