Neurotoxicant-induced elevation of adrenomedullin expression in hippocampus and glia cultures.

Adrenomedullin (AM), a vasoactive peptide first isolated from pheochromocytoma, has been reported to be present in neurons in the central nervous system and in tumors of neural and glial origin. In this study, we investigated AM expression both in the hippocampus and in glial cell cultures using a chemical-induced model of injury. An acute intraperitoneal injection of the organometal trimethyltin (TMT) results in neurodegeneration of the hippocampal CA3-4 pyramidal cell layer. Within 4 days of injection, sparse, punctate staining for AM and lectin was evident in the CA3-4 region; by 10 days, a minimal level of CA3-4 neuronal degeneration was evident, with an increase in glial fibrillary acidic protein (GFAP)-positive astrocytes throughout the hippocampus. Degeneration progressed in severity until 30 days post-TMT, with distinct positive immunoreactivity for AM in the CA4 region. mRNA levels for tumor necrosis factor (TNF)-alpha, interleukin (IL)-1alpha, GFAP, and AM in the hippocampus were increased over control levels within 4 days following TMT. In cultured glial cells, a 6 hr exposure to TMT (10 microM) produced a morphological response of the cells and increased immunoreactivity for vimentin, GFAP, and AM. mRNA levels for TNFalpha, IL-1alpha, GFAP, vimentin, and AM were elevated within 3-6 hr of exposure. In culture, neutralizing antibodies to IL-1alpha and TNFalpha were effective in inhibiting the TMT-induced elevation of AM mRNA. These data suggest an interaction between the proinflammatory cytokines and glia response in the regulation of AM in response to injury.

Pharmacology and toxicology of ethinyl estradiol and norethindrone acetate in experimental animals.

For over 30 years various combinations of synthetic estrogens and progestins have been used in oral contraceptive formulations. Ethinyl estradiol (EE) and norethindrone acetate (NA) are common synthetic hormones used in oral contraceptives such as Loestrin, Brevicon, Ortho-Novum, Norlestrin, and Norinyl. In recent years these oral contraceptives have been considered for development in other therapeutic indications. Given the use of these agents for other clinical indications with different and larger target populations, an updated comprehensive review of the toxicology literature of estrogens and progestins is warranted. This review will summarize available data on the pharmacology and toxicology of estrogens and progestins with an emphasis on the specific synthetic hormones EE and NA. Ethinyl estradiol and norethindrone acetate alone or in combination, possess low acute and chronic toxicity. In some studies, EE and/or NA increased the incidence of specific tumors in susceptible strains of rodents and dogs, but not monkeys. These agents are not teratogenic when given in combination. Alone EE and NA have clastogenic properties. Overall, the animal data demonstrates that long-term exposure to EE and NA formulations pose very little health risks to humans.

Neurodegeneration and glia response in rat hippocampus following nitro-L-arginine methyl ester (L-NAME).

Hippocampal neurodegeneration and glia response was examined following administration of the nitric oxide synthase inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME). Male Long-Evans rats received L-NAME (50 mg/kg, ip) either once or twice a day for 4 days. Both dosing schedules decreased NOS-activity by approximately 90%. At 10 and 30 days following cessation of L-NAME (2x/day), moderate neuronal death was evident in CA1-2 pyramidal cells and dentate granule cells. Neurodegeneration was accompanied by increased astrocyte glial fibrillary acidic protein (GFAP) immunoreactivity yet, minimal astrocyte hypertrophy. Microglia response was limited to an increase in ramified microglia at 10 days, returning to normal by 30 days. As early as 4 days post-dosing (2x/day), GFAP mRNA levels were significantly elevated as were mRNA levels for tumor necrosis factor-alpha (TNFalpha), interleukin-1alpha (IL-1alpha), and interleukin 6 (IL-6). No alterations were seen with L-NAME dosing limited to once a day. The co-administration of a hippocampal neurotoxicant, trimethyltin (TMT), with the last dose of L-NAME (2x/day), produced an additive response pattern of neuronal degeneration including both CA1-2 and CA3-4 pyramidal neurons accompanied by TMT-induced astrocyte hypertrophy and prominent microglia reactivity. This was preceded by elevations in mRNA levels for GFAP, TNFalpha, IL-1alpha, and IL-6 similar to those seen with each substance alone. These data suggest that high levels of L-NAME can produce a pro-inflammatory environment in the brain and that neurodegeneration and neuroglia responses in the hippocampus can be induced by an alteration in the balance and regulation of local nitric oxide levels.

Induction of tumor necrosis factor alpha in cultured glial cells by trimethyltin.

Within the central nervous system, cytokines are thought to play an active role in pathophysiological changes seen in various neurodegenerative diseases and trauma. Previous studies in our laboratory demonstrated that systemic administration of the neurotoxicant trimethyltin (TMT) produced a rapid and sustained elevation of CNS TNF alpha mRNA levels. In order to examine the effects of TMT on glial cultures in the absence of a neuronal component, primary glial cultures were exposed to TMT. Cultured glial cells undergo distinct morphological changes within 6 h of exposure to 10 microM TMT. This is characterized by an initial retraction of astrocytic processes revealing long, thin GFAP-dense processes and enlarged cell bodies, progressing to distinct retraction of plasmalemmna processes by 24 h. Prior to morphological changes, mRNA levels for the astrocyte-specific protein, glial fibrillary acidic protein (GFAP), increased within 3 h, as determined by Northern blot hybridization. Approximately a four-fold increase in TNF alpha mRNA levels was observed after 6 h as determined by competitive RT-PCR. This stimulation resulted in a 10-fold increase in the biologically active form of TNF alpha protein. These results suggest that a direct stimulation of glial cells may produce an early and critical response of the nervous system in chemical-induced neurotoxicity.

Trimethyltin increases interleukin (IL)-1 alpha, IL-6 and tumor necrosis factor alpha mRNA levels in rat hippocampus.

Within the central nervous system (CNS), cytokines are though to have active roles in pathophysiological changes seen in various neurological diseases and trauma. The present study was undertaken to examine the early response of pro-inflammatory cytokines following exposure to a specific neurotoxicant (trimethyltin; TMT). mRNA levels for interleukin (IL)-1 alpha, IL-1 beta, IL-6 and tumor necrosis factor (TNF) alpha were measured in the hippocampus of adult male Long-Evans hooded rats following an acute injection of trimethyltin hydroxide (8 mg TMT/kg body weight). At various times following exposure (6 h to 8 days), hippocampal tissues were excised and relative changes in cytokine mRNA levels were assessed by reverse transcription and polymerase chain reaction. IL-1 alpha, IL-6 and TNF alpha mRNA levels in the hippocampus increased within 6 h and remained elevated for 8 days. Quantitative analysis of mRNA transcripts revealed a two-fold increase in both IL-6 and TNF alpha within 6 h and a continued elevation of TNF alpha to 9-fold by 12 h. Within 96 h, glial fibrillary acidic protein (GFAP) mRNA levels were elevated in the hippocampus. Histological examination showed sparse individual neuronal necrosis at this time in both the pyramidal and granule cell regions with no increase in astrocyte GFAP immunoreactivity. However, an early, 24 h, response of microglial cells was indicated by increased lectin binding. This morphological profile progressed over time to a profound neuronal loss in the CA3-4 granule cell layer and marked astrocyte hypertrophy. The onset of pro-inflammatory cytokine mRNA expression appears to be temporally associated with histological evidence of elevated microglia in the hippocampus. It is proposed that microglia and pro-inflammatory cytokines play a modulatory role in the early stages of TMT-induced neurotoxicity.

Sensitivity of adenosine triphosphatases in different brain regions to polychlorinated biphenyl congeners.

Polychlorinated biphenyl (PCBs) mixtures contain a number of different congeners, some of which have been proposed to be neuroactive. Recent studies have suggested that ortho-substituted PCBs may be neuroactive, while 'dioxin-like' non-ortho-substituted congeners are not. This study compared the in vitro effects of a putative neuroactive ortho-biphenyl (2,2'-dichlorobiphenyl; DCBP) with that of a putative non-neuroactive congener lacking ortho-chlorine substitutions (3,3',4,4',5-pentachlorobiphenyl; PCBP) on Mg(2+)-ATPase activity in mitochondrial and synaptosomal preparations from striatum, hypothalamus, cerebellum and hippocampus. In these studies, DCBP significantly inhibited oligomycin-sensitive (OS) Mg(2+)-ATPase activity in all four brain regions in a concentration-dependent manner; PCBP, on the other hand, had no effect on OS Mg(2+)-ATPase activity in any brain region examined at concentrations up to 100 microM. The striatum, a dopamine-rich region, was not preferentially sensitive to the effects of DCBP. Furthermore, DCBP did not inhibit synaptosomal Na+/K(+)-ATPase activity, suggesting a specificity of action on OS Mg(2+)-ATPase. These data support previous structure-activity relationships, suggesting that ortho-substituted PCB congeners are neuroactive while non-ortho-substituted congeners are not. Disruption of mitochondrial oxidative energy production may play a role in the neuroactivity of ortho-chlorinated PCBs.

Comparative ability of various PCBs, PCDFs, and TCDD to induce cytochrome P450 1A1 and 1A2 activity following 4 weeks of treatment.

Toxic equivalency factors (TEFs) have been proposed for dibenzo-p-dioxins, dibenzofurans, and polyhalogenated biphenyls. The proposed toxic equivalency factors (TEFs), which are presently being evaluated in our laboratory, are currently used to estimate the potential health risk associated with exposure to complex mixtures containing these chemicals. In preliminary studies, equally potent doses, based on the published TEFs and relative enzyme-inducing potency, of 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran, 1,2,3,7,8-pentachlorodibenzofuran, 1,2,3,4,6,7,8,9-octachloro-dibenzofuran, 3,4,3',4'-tetrachlorobiphenyl, 2,3,4,3',4'-pentachlorobiphenyl, 3,4,5,3',4'-pentachlorobiphenyl, 2,3,4,3',4',5'-hexachlorobiphenyl, 2,3,4,5,3',4'-hexachlorobiphenyl, and 3,4,5,3',4',5'-hexachlorobiphenyl were administered to female B6C3F1 mice 5 days a week over a 4-week period. Hepatic, skin, and lung cytochrome P450 1A1 and hepatic 1A2 activities were determined for all chemicals tested and compared to those from TCDD-treated mice. These initial studies indicate that the present TEFs do not reliably predict induction potency for many of the chemicals. Furthermore, our data suggest that the relative inductive potency of these chemicals may be tissue specific and that estimates of TEFs based on hepatic ethoxyresorufin O-de-ethylase activity may not accurately reflect the potency of these chemicals in nonhepatic tissue. The TEFs proposed for the "dioxin-like" polychlorinated biphenyls (PCBs) overestimate the potency of these compounds by factors of 10-1000. The present study indicates that more experimental data are required before TEFs for PCBs should be used in regulatory decision making.

Na+/K(+)-ATPase in rat brain and erythrocytes as a possible target and marker, respectively, for neurotoxicity: studies with chlordecone, organotins and mercury compounds.

Due to the inaccessibility of human nerve tissue for direct biochemical evaluation, there appears to be a need to identify peripheral markers which will reflect toxicity to the central nervous system by relatively non-invasive means. The aim of this study was to investigate whether the enzyme Na+/K(+)-ATPase in erythrocytes could be used as a marker for effects on the same enzyme in brain tissue. The compounds chosen to test this hypothesis were the pesticide chlordecone, the organotin compounds triethyltin and tributyltin, mercuric chloride and methyl mercury. All compounds were found to inhibit in vitro Na+/K(+)-ATPase activity in rat brain (IC50s = 0.9-56 microM) and in rat erythrocytes (IC50s = 1.2-66 microM) with similar potencies. However, administration of these compounds in vivo at high doses produced no significant inhibition of either brain or erythrocyte Na+/K(+)-ATPase activity, despite observed symptoms of neurotoxicity. Dialysis experiments indicated that dissociation of the compounds by dilution during tissue preparation was not responsible for the lack of detectable in vivo inhibition. Measurements of metal concentrations in brain by atomic absorption spectrometry after in vivo administration of triethyltin, mercuric chloride and methyl mercury indicated that levels of these compounds were too low to inhibit significantly NA+/K(+)-ATPase activity. These results suggest that inhibition of Na+/K(+)-ATPase activity might not represent the mechanism responsible for the neurotoxicity of these compounds, and that erythrocyte Na+/K(+)-ATPase activity is not a useful marker for neurotoxicity following acute exposures.