J. Arunakaran

Studies on the protective role of vitamin C and E against polychlorinated biphenyl (Aroclor 1254)—induced oxidative damage in Leydig cells

Free radical production and lipid peroxidation are potentially important mediators in testicular physiology and toxicology. Polychlorinated biphenyls (PCBs) are global environmental contaminants that cause disruption of the endocrine system in human and animals. The present study was conducted to elucidate the protective role of vitamin C and E against Aroclor 1254-induced changes in Leydig cell steroidogenesis and antioxidant system. Adult male rats were dosed for 30 days with daily intraperitoneal (ip) injection of 2 mg/kg Aroclor or vehicle (corn oil). One group of rats was treated with vitamin C (100 mg/kg bw/day) while the other group was treated with vitamin E (50 mg/kg bw/day) orally, simultaneously with Aroclor 1254 for 30 days. One day after the last treatment, animals were euthanized and blood was collected for the assay of serum hormones such as luteinizing hormone (LH), thyroid stimulating hormone (TSH), prolactin (PRL), triiodothyronine (T3), thyroxine (T4), testosterone and estradiol. Testes were quickly removed and Leydig cells were isolated in aseptic condition. Purity of Leydig cells was determined by 3β-hydroxysteroid dehydrogenase (3β-HSD) staining method. Purified Leydig cells were used for quantification of cell surface LH receptors and steroidogenic enzymes such as cytochrome P450 side chain cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β- HSD). Leydig cellular enzymatic antioxidants superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), γ-glutamyl transpeptidase (γ-GT), glutathione-S-transferase (GST) and non-enzymatic antioxidants such as vitamin C and E were assayed. Lipid peroxidation (LPO) and reactive oxygen species (ROS) were also estimated in Leydig cells. Aroclor 1254 treatment significantly reduced the serum LH, TSH, PRL, T3, T4, testosterone and estradiol. In addition to this, Leydig cell surface LH receptors, activities of the steroidogenic enzymes such as cytochrome P450scc, 3β-HSD, 17β-HSD, antioxidant enzymes SOD, CAT, GPX, GR, γ-GT, GST and non-enzymatic antioxidants such as vitamin C and E were significantly diminished whereas, LPO and ROS were markedly elevated. However, the simultaneous administration of vitamin C and E in Aroclor 1254 exposed rats resulted a significant restoration of all the above-mentioned parameters to the control level. These observations suggest that vitamin C and E have ameliorative role against adverse effects of PCB on Leydig cell steroidogenesis.

Induction of apoptosis in human breast cancer cells by nimbolide through extrinsic and intrinsic pathway.

We aimed to investigate the cytotoxic effects of nimbolide, a limonoid present in leaves and flowers of the neem tree (Azadirachta indica) on human breast cancer cells. The molecular mechanisms involved in the apoptotic activity exerted by nimbolide were studied on the estrogen dependent (MCF-7) and estrogen independent (MDA-MB-231) human breast cancer cell lines. The growth inhibitory effect of nimbolide was assessed by MTT assay. Apoptosis induction by nimbolide treatment was determined by JC-1 mitochondrial membrane potential staining, cytochrome c release, caspase activation, cleavage of PARP and AO/EtBr dual staining. The modulation of apoptotic proteins (intrinsic pathway: Bax, bad, Bcl-2, Bcl-xL, Mcl-1, XIAP-1 and caspase-3, 9; extrinsic pathway: TRAIL, FasL, FADDR and Caspase-8) were studied by western blot and real time PCR analysis. Treatment with nimbolide resulted in dose and time-dependent inhibition of growth of MCF-7 and MDA-MB-231 cells. The occurrence of apoptosis in these cells was indicated by JC-1 staining, modulation of both intrinsic and extrinsic apoptotic signaling molecules expression and further apoptosis was confirmed by AO/EtBr dual staining. These events were associated with: increased levels of proapoptotic proteins Bax, Bad, Fas-L, TRAIL, FADDR, cytochrome c and reduced levels of the anti-apoptotic proteins Bcl-2, Bcl-xL, Mcl-1 and XIAP-1. Nimbolide induces the cleavage of pro-caspase-8, pro-caspase-3 and PARP. The above data suggest that nimbolide induces apoptosis by both the intrinsic and extrinsic pathways. With evidence of above data it is suggested that nimbolide exhibit anticancer effect through its apoptosis-inducing property. Thus, nimbolide raises new hope for its use in anticancer therapy.

Polychlorinated biphenyl (Aroclor 1254) inhibits testosterone biosynthesis and antioxidant enzymes in cultured rat Leydig cells

Polychlorinated biphenyls (PCBs) are environmental contaminants that in humans and animals disturb normal endocrine functions including gonadal functions. The present studies were aimed at determining the direct effects of PCB on Leydig cell testosterone production and antioxidant system in vitro. Adult Leydig cells were purified by Percoll gradient centrifugation method and the purity of Leydig cells was also determined by 3beta-hydroxysteroid dehydrogenase (3beta-HSD) staining method. Purified Leydig cells were exposed to different concentrations (10(-10) to 10(-7) M) of PCB (Aroclor 1254) for 6 and 12 h under basal and LH-stimulated conditions. After incubation, the cultured media were collected and used for the assay of testosterone. The treated cells were used for quantification of cell surface LH receptors and activity of steroidogenic enzymes such as cytochrome P450 side chain cleavage enzyme (P450scc), 3beta-HSD and 17beta-hydroxysteroid dehydrogenase (17beta-HSD). In addition, Leydig cellular enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), gamma-glutamyl transpeptidase (gamma-GT), glutathione-S-transferase (GST) and non-enzymatic antioxidants such as vitamin C and E were assayed. Lipid peroxidation (LPO) and reactive oxygen species (ROS) were also estimated in Leydig cells. The results indicated that Aroclor 1254 (10(-8) and 10(-7) M) treatments significantly inhibit basal and LH-stimulated testosterone production. In addition to this, the activity of steroidogenic enzymes, enzymatic and non-enzymatic antioxidants were significantly diminished in a dose- and time-dependent manner. Moreover, the LPO and ROS were elevated in a dose- and time-dependent manner under basal and LH-stimulated conditions. These findings suggest that PCBs can act directly on Leydig cells to inhibit testosterone biosynthesis by reducing steroidogenic enzymes, enzymatic and non-enzymatic antioxidants.

Quercetin inhibits invasion, migration and signalling molecules involved in cell survival and proliferation of prostate cancer cell line (PC-3)

Urokinase‐type plasminogen activator (uPA) is a serine protease that is involved in cancer progression, especially invasion and metastasis including prostate cancer. uPA activation is mediated by transactivation of uPAR and epidermal growth factor receptor (EGF‐R) in prostate cancer progression. Prostate cancer (PC‐3) cells have highly invasive capacity and they express uPA and uPAR gene. PC‐3 cells are treated with quercetin, which inhibits invasion and migration of PC‐3 cells. Quercetin downregulates uPA, uPAR and EGF, EGF‐R mRNA expressions. Quercetin inhibits cell survival factor β‐catenin, NF‐κB and also proliferative signalling molecules such as p‐EGF‐R, N‐Ras, Raf‐1, c.Fos c.Jun and p‐c.Jun protein expressions. But quercetin increased p38 mitogen‐activated protein kinase protein expression. Our results suggest that quercetin inhibit migration and invasion of prostate cancer cells. It shows the value for treatment of invasive and metastasis type of prostate cancer. Copyright

Impact of polychlorinated biphenyl Aroclor 1254 on testicular antioxidant system in adult rats.

To clarify the reproductive toxicity of polychlorinated biphenyl compounds through determination of testicular lipid peroxidation, reactive oxygen species and enzymatic and non-enzymatic antioxidants in rats exposed to Aroclor 1254. Adult male rats were administered Aroclor 1254 at a dose of 2 mg/kg per day ip for 30 days. The rats were sacrificed 24 hours after last dosing and the serum and other tissues collected and processed for relevant determinations. The body weight and the weights of the testis, epididymis, ventral prostate and seminal vesicle and the serum testosterone and estradiol were significantly decreased in Aroclor 1254 treated rats. The testicular lipid peroxidation, hydrogen peroxide and hydroxyl radical were significantly elevated whereas, testicular antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione-S-transferase (GST) and glutathione reductase (GR) were significantly decreased. The non-enzymatic antioxidants, vitamin C and vitamin E, were also decreased. These results suggest that Aroclor 1254 induces an increase in the lipid peroxidation, hydrogen peroxide and hydroxyl radical and diminish in the antioxidant defense system in rats, indicating that the free radical-dependent mechanism may play an important role in the testicular toxicity of polychlorinated biphenyls.

Quercetin reverses EGF-induced epithelial to mesenchymal transition and invasiveness in prostate cancer (PC-3) cell line via EGFR/PI3K/Akt pathway

Epidermal growth factor (EGF) plays an important role in metastasis and tumorigenesis of prostate cancer. Epithelial-mesenchymal transition (EMT) is a process in tumor progression during which cancer cells undergo dramatic changes acquiring highly invasive properties. The purpose of this study was to determine the effect of quercetin on EGF-induced EMT in prostate cancer (PC-3) cell line. Quercetin, a plant flavonoid, prevented EGF-induced invasion and migration of PC-3 cells. The protein and mRNA expressions of E-cadherin and N-cadherin were studied by immunocytochemistry, Western blotting and real-time polymerase chain reaction. Quercetin prevented EGF-induced expression of N-cadherin and vimentin and increased the expression of E-cadherin in PC-3 cells, therefore preventing EGF-induced EMT. EGF-induced cell adhesion proteins, intercellular adhesion molecule and vascular cell adhesion molecule were significantly decreased by quercetin treatment. Furthermore, mRNA and protein expressions of Snail, Slug and Twist showed that quercetin significantly decreased EGF-induced expressions of Snail, Slug and Twist. The protein expressions of epidermal growth factor receptor (EGFR)/phosphatidylinositide 3-kinases (PI3K)/Akt/extracellular signal-regulated kinase (ERK)1/2 pathway showed that quercetin prevents EGF-induced EMT via EGFR/PI3k/Akt/ERK1/2 pathway and by suppressing transcriptional repressors Snail, Slug and Twist in PC-3 cells. Thus, it is concluded from the present study that quercetin may prevent cancer metastasis by targeting EMT.

Quercetin regulates insulin like growth factor signaling and induces intrinsic and extrinsic pathway mediated apoptosis in androgen independent prostate cancer cells (PC-3)

Progression of prostate cancer is facilitated by growth factors that activate critical signaling cascades thereby promote prostate cancer cell growth, survival, and migration. To investigate the effect of quercetin on insulin-like growth factor signaling and apoptosis in androgen independent prostate cancer cells (PC-3), IGF-IR, PI-3K, p-Akt, Akt, cyclin D1, Bad, cytochrome c, PARP, caspases-9 and 10 protein levels were assessed by western blot analysis. Mitochondrial membrane potency was detected by rhodamine-123 staining. Quercetin induced caspase-3 activity assay was performed for activation of apoptosis. Further, RT-PCR was also performed for Bad, IGF-I, II, IR, and IGFBP-3 mRNA expression. Quercetin significantly increases the proapoptotic mRNA levels of Bad, IGFBP-3 and protein levels of Bad, cytochrome C, cleaved caspase-9, caspase-10, cleaved PARP and caspase-3 activity in PC-3 cells. IGF-IRβ, PI3K, p-Akt, and cyclin D1 protein expression and mRNA levels of IGF-I, II and IGF-IR were decreased significantly. Further, treatment with PI3K inhibitor (LY294002) and quercetin showed decreased p-Akt levels. Apoptosis is confirmed by loss of mitochondrial membrane potential in quercetin treated PC-3 cells. This study suggests that quercetin decreases the survival of androgen independent prostate cancer cells by modulating the expression of insulin-like growth factors (IGF) system components, signaling molecules and induces apoptosis, which could be very useful for the androgen independent prostate cancer treatment.

Protective role of melatonin on PCB (Aroclor 1254) induced oxidative stress and changes in acetylcholine esterase and membrane bound ATPases in cerebellum, cerebral cortex and hippocampus of adult rat brain

Polychlorinated biphenyls (PCBs) are one of the environmental toxicants and neurotoxic compounds which induce the production of free radicals leading to oxidative stress. Membrane proteins that control ion gradients across organellar and plasma membranes appear to be particularly susceptible to oxidation induced changes. Melatonin plays an important role in neurodegenerative diseases as an antioxidant and neuroprotector. The aim of this study was to determine the protective role of melatonin on PCB (Aroclor 1254) induced changes in activities of membrane bound ATPases and acetylcholine esterase in selected brain regions of adult rats. Group I: rats intraperitoneally (i.p.) administered corn oil (vehicle) for 30 days. Group II: rats injected i.p. with Aroclor 1254 (PCB) at 2 mg/kg bw/day for 30 days. Groups III and IV: rats intraperitoneally received melatonin (5 or 10 mg/kg bw/day) simultaneously with Aroclor 1254 for 30 days. Groups V and VI: rats intraperitoneally received melatonin (5 or 10 mg/kg bw/day) alone for 30 days. After 30 days, rats were sacrificed and the brain regions were dissected to cerebral cortex (Cc), cerebellum (C) and hippocampus (H). Lipid peroxidation (LPO), hydrogen peroxide (H2O2), hydroxyl radical (*OH) and the activities of Na+K+ ATPase, Ca2+ ATPase, Mg2+ ATPase and acetyl cholinesterase were determined. Reduced glutathione (GSH) level was also determined. Melatonin levels in serum was measured by enzyme labeled immunosorbent assay (ELISA). Activities of all the enzymes and GSH level were decreased while an increase in H2O2, *OH and LPO were observed in brain regions of PCB treated animals. Melatonin levels in serum was decreased in PCB exposed animals. Exogenous melatonin supplementation retrieved all the parameters, significantly. These results suggest that PCB alters membrane bound ATPases and cholinergic function by inducing oxidative stress in brain regions, which can be protected by melatonin.

Effects of piperine on testis of albino rats.

Piperine was administered to mature male albino rats at doses of 5 and 10 mg/kg body weight, p.o., respectively, for 30 days. Only a 10 mg dose of piperine treatment caused a significant reduction in the weights of testis and accessory sex organs. Histological studies revealed that piperine at a 5 mg dose caused partial degeneration of germ cell types, whereas at a 10 mg dose, it caused severe damage to the seminiferous tubule, decrease in seminiferous tubular and Leydig cell nuclear diameter and desquamation of spermatocytes and spermatids. Correlated to the structural changes, a fall in caput and cauda epididymal sperm concentrations was also evident. A 10 mg dose of piperine also caused a marked increase in serum gonadotropins and a decrease in intratesticular testosterone concentration, despite normal serum testosterone titres.

Effect of melatonin on PCB (Aroclor 1254) induced neuronal damage and changes in Cu/Zn superoxide dismutase and glutathione peroxidase-4 mRNA expression in cerebral cortex, cerebellum and hippocampus of adult rats

Polychlorinated biphenyls (PCBs) are one of the environmental toxicants and neurotoxic compounds which induce the production of free radicals leading to oxidative stress. Free radicals represent a class of biologically generated species that pose a potential threat to neuronal survival. Cu/Zn superoxide dismutase (SOD) and glutathione peroxidase-4 (GPx-4) are the key cellular antioxidant enzymes by which neurons and other cells detoxify free radicals and protect themselves from damage. Melatonin, an indoleamine plays an important role in neurodegenerative diseases as an antioxidant and neuroprotector. The aim was to carry out to investigate the effect of melatonin on PCB (Aroclor 1254) induced changes in histomorphology and Cu/Zn SOD, GPx-4 mRNA expression in selected brain regions of adult rats. Group I: rats intraperitoneally (i.p.) administered with corn oil (vehicle) for 30 days. Group II: rats injected (i.p.) with Aroclor 1254 (PCB) at 2mg/kgbw/day for 30 days. Groups III and IV: rats (i.p.) received melatonin (5 or 10mg/kgbw/day) simultaneously with PCB for 30 days. Groups V and VI: rats (i.p.) received melatonin (5 or 10mg/kgbw/day) alone for 30 days. After 30 days, rats were sacrificed and the brain regions were dissected to cerebral cortex, cerebellum and hippocampus. Activities of enzymatic antioxidants such as total SOD, Cu/Zn SOD, Mn SOD, glutathione peroxidase (GPx) were estimated. mRNA expressions of Cu/Zn SOD and GPx-4 were quantified by reverse transcriptase polymerase chain reaction (RT-PCR) method. Histological study was also observed. Specific activities of all antioxidant enzymes and mRNA expression of Cu/Zn SOD and GPx-4 were decreased in brain regions of PCB exposed animals. Neuronal damages were observed in all the brain regions. Exogenous melatonin supplementation retrieved all the parameters. These results suggest that melatonin protects PCB-induced oxidative stress and prevents neuronal damage in brain regions.