Alterations of thyroidal status in brain regions and hypothalamo-pituitary-blood-thyroid-axis associated with dopaminergic depletion in substantia nigra and ROS formation in different brain regions after MPTP treatment in adult male mice.

MPTP produces oxidative stress, damages niagrostriatal dopaminergic neurons and develops Parkinsonism in rodents. Due to paucity of information, the thyroidal status in brain regions and peripheral tissues during different post-treatment days in MPTP-induced mice had been executed in the present study. MPTP depleted tyrosine hydroxylase protein expressions that signify the dopaminergic neuronal damage in substantia nigra. MPTP elevated ROS formation differentially in brain regions (cerebral cortex, hippocampus, substantia nigra) with maximal elevation at hippocampus. The changes in thyroid hormone (T4 and T3) levels indicate that brain regions might combat the adverse situation by keeping the levels of thyroid hormones either unchanged or in the elevated conditions in the latter phases (day-3 and day-7), apart from the depletion of thyroid hormones in certain brain regions (T4 in SN and hippocampus, T3 in hippocampus) as the immediate (day-1) effects after MPTP treatment. MPTP caused alterations of cellular morphology, RNA:Protein ratio and TPO protein expression, concomitantly depleted TPO mRNA expression and elevated TSH levels in the thyroid gland. Although T4 levels changed differentially, T3 levels remained unaltered in thyroid gland throughout the post-treatment days. Results have been discussed mentioning the putative role of T4 and TSH in apoptosis and/or proliferation/differentiation of thyrocytes. In blood, T4 levels remained unchanged while the changes in T3 and TSH levels did not signify the clinical feature of hypo/hyperthyroidism of animals. In the pituitary, both T4 and T3 levels remained elevated where TSH differentially altered (elevated followed by depletion) during post-treatment days. Notably, T4, T3 and TSH levels did not alter in hypothalamus except initial (day-1) depletion of the T4 level. Therefore, the feedback control mechanism of hypothalamo-pituitary-blood-thyroid-axis failed to occur after MPTP treatment. Overall, MPTP altered thyroidal status in the brain and peripheral tissues while both events might occur in isolation as well.

Alteration in Nuclear Factor-KappaB Pathway and Functionality of Estrogen via Receptors Promote Neuroinflammation in Frontal Cortex after 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine Treatment.

The MPTP mediated neurodegeneration in substantia nigra has been well studied, but not the status of frontal cortex. The novelty of the present study is to explore the sex difference of frontal cortex during MPTP intoxication and to investigate the role of estrogen and its receptors in presence of glial cells in a time chase experiment; to identify which pathway of NF-kappaB exist to proceed the neuroinflammation; to investigate the estrogen binding with its nuclear or cytosolic receptors and whether any direct relation exists between estrogen receptor (ER) -beta and NF-kappaB molecules p65 and RelB. The progression of neurodegeneration occurred with the association of glial cells and functional (via its nuclear and cytosolic receptors) estrogen level. Both the canonical and/or non canonical pathways of NF-kappaB exist in frontal cortex of both the sexes after MPTP treatment. The homodimeric or heterodimeric form of ER-beta binds with NF-kappaB molecules p65 and RelB differently, but the canonical or non canonical pathways of NF-kappaB molecules could not be stopped or may be promoted. The changes in the molecular and cellular pattern in frontal cortex of both sexes during MPTP intoxication depends on the estrogen function via its nuclear or cytosolic estrogen receptors.

Copper-induced immunotoxicity involves cell cycle arrest and cell death in the liver.

Inorganic copper, such as that in drinking water and copper supplements, largely bypasses the liver and enters the free copper pool of the blood directly and that promote immunosuppression. According to our previous in vivo report, we evaluate the details of the apoptotic mechanism in liver, we have investigated how copper regulates apoptotic pathways in liver. We have analyzed different protein expression by Western blotting and immunohistochemistry expression. We have also have measured mitochondrial trans-membrane potential, Annexin V assay, ROS, and CD4(+) and CD8(+) population in hepatocyte cells by flow cytometry. Copper-treated mice evidenced immunotoxicity as indicated by dose-related, distinct histomorphological changes in liver. Flow cytometric analyses revealed a dose-related increase in the percentages of hepatocyte cells in the Sub-G0/G1 state, further confirmed by Annexin V binding assay. In addition, the copper treatments altered the expression of apoptotic markers, further ROS generation and mitochondrial trans-membrane potential changes promote intrinsic pathway of apoptosis that was p53 independent. Apart from the role of inflammation, our findings also have identified the role of other partially responsible apoptotic molecules p73 that differentially changed due to copper treatment. Our study demonstrates how apoptotic pathways regulate copper-induced immunosuppression in liver.

Copper induced immunotoxicity promote differential apoptotic pathways in spleen and thymus.

Inorganic copper, such as that in drinking water and copper supplements, largely bypasses the liver and enters the free copper pool of the blood directly and that promote immunosuppression. Nevertheless, the signaling pathways underlying copper-induced immune cell death remains largely unclear. According to our previous in vivo report, to evaluate the further details of the apoptotic mechanism, we have investigated how copper regulates apoptotic pathways in spleen and thymus. We have analyzed different protein expression by western blotting and immunohistochemistry and mRNA expression by RT-PCR and gel electrophoresis. We also have measured mitochondrial trans-membrane potential, ROS and CD4(+) and CD8(+) population by flow cytometry. Sub lethal doses of copper in spleen and thymus of in vivo Swiss albino mice promote different apoptotic pathways. In case of spleen, ROS generation and mitochondrial trans-membrane potential changes promotes intrinsic pathway of apoptosis that was p53 independent, ultimately leads to decrease in CD4(+) T cell population and increase in CD8(+) T cell population. However in case of thymus, ROS generation and mitochondrial trans-membrane potential changes lead to death receptor that regulate extrinsic and intrinsic pathways of apoptosis and the apoptotic mechanism which was p53 dependent. Due to copper treatment, thymic CD4(+) T cell population decreased and CD8(+) T cell population was increased or proliferated. Apart from the role of inflammation, our findings also have identified the role of other partially responsible apoptotic molecules like p27, p73, p62, poly (ADP-ribose) polymerase (PARP) that differentially changed due to copper treatment in spleen and thymus of Swiss albino mice. Present study firstly demonstrates how apoptotic pathways differentially regulate copper induced immunosuppression.

Copper-induced immunotoxicity involves cell cycle arrest and cell death in the spleen and thymus.

Copper is an essential trace element for human physiological processes. To evaluate the potential adverse health impact/immunotoxicological effects of this metal in situ due to over exposure, Swiss albino mice were treated (via intraperitoneal injections) with copper (II) chloride (copper chloride) at doses of 0, 5, or 7.5 mg copper chloride/kg body weight (b.w.) twice a week for 4 wk; these values were derived from LD50 studies using copper chloride doses that ranged from 0 to 40 mg/kg BW (2×/wk, for 4 wk). Copper treated mice evidenced immunotoxicity as indicated by dose-related decreases and increases, respectively, in thymic and splenic weights. Histomorphological changes evidenced in these organs were thymic atrophy, white pulp shrinkage in the spleen, and apoptosis of splenocytes and thymocytes; these observations were confirmed by microscopic analyses. Cell count analyses indicated that the proliferative functions of the splenocytes and thymocytes were also altered because of the copper exposures. Among both cell types from the copper treated hosts, flow cytometric analyses revealed a dose related increase in the percentages of cells in the Sub-G0/G1 state, indicative of apoptosis which was further confirmed by Annexin V binding assay. In addition, the copper treatments altered the expression of selected cell death related genes such as EndoG and Bax in a dose related manner. Immunohistochemical analyses revealed that there was also increased ubiquitin expression in both the cell types. In conclusion, these studies show that sublethal exposure to copper (as copper chloride) induces toxicity in the thymus and spleen, and increased Sub G0/G1 population among splenocytes and thymocytes that is mediated, in part, by the EndoG-Bax-ubiquitin pathway. This latter damage to these cells that reside in critical immune system organs are likely to be important contributing factors underlying the immunosuppression that has been documented by other investigators following acute high dose/chronic low-medium dose exposures to copper agents.

Differential regional expression patterns of α-synuclein, TNF-α, and IL-1ß; and variable status of dopaminergic neurotoxicity in mouse brain after Paraquat treatment.

BACKGROUND: Paraquat (1, 1-dimethyl-4, 4-bipyridium dichloride; PQ) causes neurotoxicity, especially dopaminergic neurotoxicity, and is a supposed risk factor for Parkinson's disease (PD). However, the cellular and molecular mechanisms of PQ-induced neurodegeneration are far from clear. Previous studies have shown that PQ induces neuroinflammation and dopaminergic cell loss, but the prime cause of those events is still in debate. METHODS: We examined the neuropathological effects of PQ not only in substantia nigra (SN) but also in frontal cortex (FC) and hippocampus of the progressive mouse (adult Swiss albino) model of PD-like neurodegeneration, using immunohistochemistry, western blots, and histological and biochemical analyses. RESULTS: PQ caused differential patterns of changes in cellular morphology and expression of proteins related to PD and neuroinflammation in the three regions examined (SN, FC and hippocampus). Coincident with behavioral impairment and brain-specific ROS generation, there was differential immunolocalization and decreased expression levels of tyrosine hydroxylase (TH) in the three regions, whereas α-synuclein immunopositivity increased in hippocampus, increased in FC and decreased in SN. PQ-induced neuroinflammation was characterized by area-specific changes in localization and appearances of microglial cells with or without activation and increment in expression patterns of tumor necrosis factor-α in the three regions of mouse brain. Expression of interleukin-1ß was increased in FC and hippocampus but not significantly changed in SN. CONCLUSION: The present study demonstrates that PQ induces ROS production and differential α-synuclein expression that promotes neuroinflammation in microglia-dependent or -independent manners, and produces different patterns of dopaminergic neurotoxicity in three different regions of mouse brain.