Prenatal exposure to hexavalent chromium disrupts testicular steroidogenic pathway in peripubertal F1 rats.

We have reported sub-fertility in F1 progeny rats with gestational exposure to hexavalent chromium [Cr(VI)], which had disrupted Sertoli cell (SC) structure and function, and decreased testosterone (T). However, the underlying mechanism for reduced T remains to be understood. We tested the hypothesis "transient prenatal exposure to Cr(VI) affects testicular steroidogenesis by altering hormone receptors and steroidogenic enzyme proteins in Leydig cells (LCs)." Pregnant Wistar rats were given drinking water containing 50, 100, and 200 mg/L potassium dichromate during gestational days 9-14, encompassing fetal differentiation window of the testis from the bipotential gonad. F1 male rats were euthanized on postnatal day 60 (peripubertal rats with adult-type LCs alone). Results showed that prenatal exposure to Cr(VI): (i) increased accumulation of Cr(III) in the testis of F1 rats; (ii) increased serum levels of luteinizing and follicle stimulating hormones (LH and FSH), and 17ß estradiol, and decreased prolactin and T; (iii) decreased steroidogenic acute regulatory protein, cytochrome P450 11A1, cytochrome P450 17A1, 3ß- and 17ß-hydroxysteroid dehydrogenases, cytochrome P450 aromatase and 5α reductase proteins, (iv) decreased specific activities of 3ß and 17ß hydroxysteroid dehydrogenases; (v) decreased receptors of LH, androgen and estrogen in LCs; (vi) decreased 5α reductase and receptor proteins of FSH, androgen, and estrogen in SCs. The current study concludes that prenatal exposure to Cr(VI) disrupts testicular steroidogenesis in F1 progeny by repressing hormone receptors and key proteins of the steroidogenic pathway in LCs and SCs.

JNK1 and JNK3 play a significant role in both neuronal apoptosis and necrosis. Evaluation based on in vitro approach using tert-butylhydroperoxide induced oxidative stress in neuro-2A cells and perturbation through 3-aminobenzamide.

In spinal cord injury (SCI), oxidative stress in the penumbra of the injury site is a characteristic feature. The predominance of necrosis over apoptosis in the ensuing delayed cell death results in progressive waves of necrosis affecting neighboring cells and thus exaggerates the severity of the lesion. Necrosis has been classified into subtypes based on the active molecular players and parthanatos is one among them, which is characterized by the over activation of PARP1 as the pre-mitochondrial event that triggers necrosis. Parthanatos being the necrosis mode reported in SCI, we intended to study the molecular players in the elusive pre-mitochondrial events of PARP1 over activation using an in vitro model. tert-Butylhydroperoxide (tBuOOH) was reported to induce oxidative stress in various cell types including Neuro-2A cells. Using a tailored protocol, a predominantly PARP1 mediated necrotic mode of cell death was obtained in Neuro-2A cells using tBuOOH. By perturbing the progress of necrosis using 3-amniobenzamide, a known PARP1 inhibitor, it was found that JNK1 and JNK3 but not JNK2 were involved in pre-mitochondrial stages of PARP1 mediated cell death. Given that JNK1 and JNK3 play a role in apoptosis also, they may serve as common targets to counter both apoptosis and necrosis. The in vitro model used in the present study may be useful in delineating molecular mechanisms in necrosis.

Male reproductive toxicity of CrVI: In-utero exposure to CrVI at the critical window of testis differentiation represses the expression of Sertoli cell tight junction proteins and hormone receptors in adult F1 progeny rats.

The effect of gestational exposure to CrVI (occupational/environmental pollutant and target to Sertoli cells(SC)) was tested in a rat model during the testicular differentiation from the bipotential gonad may interrupt spermatogenesis by disrupting SC tight junctions(TJ) and it's proteins and hormone receptors. Pregnant Wistar rats were exposed to 50/100/200ppm CrVI through drinking water during embryonic days 9-14. On Postnatal day 120, testes were subjected to ion exchange chromatographic analysis and revealed increased level of CrIII in SCs and germ cells, serum and testicular interstitial fluid(TIF). Microscopic analyses showed seminiferous tubules atrophy and disruption of SC TJ, which also recorded decreased testosterone in TIF. mRNA and Protein expression analyses attested decreased level of Fshr, Ar, occludin and claudin-11 in SCs. Immunofluorescent detection revealed weak signal of TJ proteins. Taken together, we concluded that gestational exposure to CrVI interferes with the expression of SC TJ proteins due to attenuated expression of hormone receptors.

Expression of adenosine receptors and vegf during angiogenesis and its inhibition by pentoxifylline-A study using zebrafish model.

Angiogenesis, formation of new blood vessels is an important process involved in neovascular diseases and tumor progression. Understanding and defining novel therapeutic targets of neovascular diseases like retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration have been hindered by a lack of appropriate animal models. Zebrafish provides an excellent vertebrate model to study above disorders since its circulatory system and retinal layers are similar to mammals. Adenosine is a known mediator of angiogenesis in hypoxic condition and adenosine receptor antagonists such as theophylline, theobromine are known to exert antiangiogenic properties. We evaluated the anti-angiogenic potential of a methylxanthine pentoxifylline (PTX) with various concentrations (0.1-1mM) at 50% epiboly stage (5.2 hpf) of zebrafish embryos and studied the mRNA expression of major angiogenic factors like vegfaa and its receptors under normal conditions and when treated with an adenosine analog NECA (5'-N-ethylcarboxamidoadenosine). Upregulation of adenosine receptors, hif-1α and vegfaa by NECA could possibly mimic hypoxic condition, but PTX downregulated vegfaa and other growth factors at 1mM concentration. Vegfa protein expression was also downregulated by PTX in the retina and the compound did not damage the retinal cells. Embryos treated with PTX generated abnormal phenotypic variants with poor vasculature, tail bending and developmental delay at 1mM. Survival rates, heart rate and hatching rates were also significantly lower. Targeting the vegf signaling pathway with small molecules inhibiting adenosine receptors in addition to antagonizing vegf might be a promising approach to treat neovascular diseases of the retina and also tumors.

Contusive spinal cord injury up regulates mu-opioid receptor (mor) gene expression in the brain and down regulates its expression in the spinal cord: possible implications in spinal cord injury research.

Traumatic spinal cord injury (SCI) is one of the dreaded neurological conditions and finding a cure for it has been a hot area of research. Naloxone - a mu-opiate receptor (mor) antagonist was considered for SCI treatment based on its positive effects under shock conditions. In contrary to animal studies based reports about the potential benefits of naloxone in treating SCI, a large scale clinical trial [National Acute Spinal Cord Injury Study II (NASCIS II)] conducted in USA failed to witness any effectiveness. The inconsistency noticed was intriguing. Therefore, the objective of the present study was to re-examine the role of naloxone in treating SCI using a highly standardised Multicenter Animal Spinal Cord Injury Study (MASCIS) animal model of contusive SCI. Results indicated that naloxone produced negligible and insignificant neuroprotection. In an attempt to understand the cause for the failure, it was found that mu-opioid receptor (mor) gene expression was upregulated in the brain but was down regulated in the spinal cord after contusive SCI. Given that the beneficial effects of naloxone are through its action on the mor, the results indicate that unlike the brain, spinal cord might not be bracing to utilise the opiate system in the repair process. This could possibly explain the failure of naloxone treatment in NASCIS II. To conclude, opiate antagonists like naloxone may be neuroprotective for treating traumatic brain injuries, but not for traumatic/contusive spinal cord injuries.

Neuroprotective role of naringenin on carbaryl induced neurotoxicity in mouse neuroblastoma cells.

OBJECTIVE: Neuroprotective effect of naringenin against carbaryl toxicity was studied in mouse neuroblastoma cell line. MATERIALS AND METHODS: Mouse neuroblastoma cells (Neuro 2A) obtained from National Center for Cell Sciences, Pune, India were either exposed to carbaryl or pre-treated with naringenin (a flavonoid prepared from grape fruit) before their exposure to carbaryl. Results were analyzed using MTT [3-4,5-Dimethylthiazol-2-yl)-2,5-diphenltetrazolium bromide] assay for cell viability, FACS (fluorescence assisted cell sorting) analysis for apoptotic and necrotic cell populations, DCFH-DA (2`,7`-dichlorofluorescin-diacetate) assay for Reactive Oxygen Species (ROS) visualization, JC-1 staining for determining mitochondrial membrane potential and real-time PCR for quantifying pro and anti-apoptotic gene expression. RESULTS: Exposure to naringenin resulted in better survival of Neuro 2A cells which were subsequently subjected to carbaryl toxicity. Treatment with naringenin was found to reduce the oxidative stress by decreasing the ROS and was found to maintain the integrity of mitochondrial membrane potential. It was also found to downregulate pro-apoptotic genes (BAX and Caspase-3) while upregulating anti-apototic gene (Bcl2). CONCLUSION: The results of this pilot study underline the potential of naringenin in treating carbaryl induced neurotoxicity and further studies are warranted to establish the effect of naringenin in vivo conditions.