Effect of cadmium-polluted water on plasma levels of tumor necrosis factor-α, interleukin-6 and oxidative status biomarkers in rats: Protective effect of curcumin

The present study was designed to investigate the effect of CdCl₂-polluted drinking water (40 mg CdCl₂/L) on the level of TNF-α and IL-6, as well as oxidative status biomarkers in plasma of rats. The possible protective effect of oral administration of curcumin (50 mg/kg body weight/day) was assessed. Results illustrated that Cd exposure significantly elevated the plasma levels of TNF-α and IL-6 (p < 0.001) as compared to normal rats. Also, Cd administration resulted in a significant elevation in the lipid peroxidation and markedly reduction in the activities of SOD and catalase as well as the level of glutathione and total antioxidant capacity in plasma. The co-treatment of Cd with curcumin significantly reduced the levels of TNF-α and IL-6 and ameliorated the alteration in oxidative status biomarkers induced by Cd. Negative correlation between IL-6 or TNF-α was and the plasma activities of catalase, SOD and the level of total antioxidant capacity were found in rats exposed to Cd. Conclusion: Cadmium toxicity induced the release of TNF-α and IL-6 which is associated with systemic oxidative stress. This may be involved in the mechanism of the Cd toxicity. On the other hand, the findings suggest the curative action of curcumin against Cd toxicity.     

Contribution of equilibrative nucleoside transporter (ENT) 2 to fluorouracil transport in rat placental trophoblast cells

Fluorouracil is used for treatment of breast cancer even in pregnant women, except during fetal organogenesis. The purpose of this study was to clarify the transport mechanism of fluorouracil at the rat placental barrier. Maternal-to-fetal transfer of [³H]fluorouracil in rats at gestational day 19.5 was saturable and much higher than that of [¹⁴C]sucrose. The uptake of [³H]fluorouracil was also saturable in rat placental trophoblast TR-TBT 18d-1 cells, which express both equilibrative nucleoside transporter (ENT) 1 and ENT2. Nitrobenzylthioinosine (NBMPR) at 0.1 μM had no effect on [³H]fluorouracil uptake by TR-TBT 18d-1 cells, but 100 μM NBMPR almost completely inhibited the saturable component, suggesting involvement of ENT2, rather than ENT1 in the transport. Rat ENT2 cRNA-injected oocytes showed significantly increased [³H]fluorouracil uptake compared with water-injected oocytes, while rat ENT1 cRNA-injected oocytes did not show an increase of [³H]fluorouracil uptake. The Michaelis–Menten constant for rat ENT2-mediated uptake of [³H]fluorouracil was 4.21 mM. The expression profile of ENT2 mRNA in rat placenta during pregnancy was almost constant from 13.5 to 21.5 days of gestation. In conclusion, ENT2 appears to be the mediator of fluorouracil transport in rat placental trophoblast cells.     

Exposure to 2,4-dichlorophenoxyacetic acid alters glucose metabolism in immature rat Sertoli cells

The purpose of this study was to determine the effects of 2,4-D, an herbicide used worldwide also known as endocrine disruptor, in Sertoli cell (SC) metabolism.Immature rat SCs were maintained 50 h under basal conditions or exposed to 2,4-D (100 nM, 10 μM and 1 mM). SCs exposed to 10 μM and 1 mM of 2,4-D presented lower intracellular glucose and lactate content. Exposure to 10 μM of 2,4-D induced a significant decrease in glucose transporter-3 mRNA levels and phosphofructokinase-1 mRNA levels decreased in cells exposed to 100 nM and 10 μM of 2,4-D. Exposure to 100 nM and 10 μM also induced a decrease in lactate dehydrogenase (LDH) mRNA levels while the LDH protein levels were only decreased in cells exposed to 1 mM of 2,4-D.Exposure to 2,4-D altered glucose uptake and metabolization in SCs, as well as lactate metabolism and export that may result in impaired spermatogenesis.     

Subchronic toxicity study of ulvan from Ulva pertusa (Chlorophyta) in Wistar rats

Ulvan extracted from Ulva pertusa (Chlorophyta) is a group of sulfated heteropolysaccharide, for simplicity, the sulfated polysaccharide is referred to as ulvan in this paper. To our knowledge, there is no detailed report investigating the toxicity of ulvan. In this study, the subchronic (6 months) toxicity of varying levels of ulvan extracted from U. pertusa was investigated in Wistar rats after oral administration. ALT, ALB, ALP, WBC, PLT, and liver relative organ weigh of female rats showed significantly difference at 3000 mg/kg body weight per day, compared with control group. On the other hand, TG, T-CHO concentrations of female rats (6 months) were significantly decreased at 600, 1200 and 3000 mg/kg body weight per day. This result proved that ulvan had antihyperlipidemic activity. Beside, ulvan showed anticoagulant activity in this study. Overall, our findings indicated that ulvan had affected specific hematology, serum biochemistry parameters and liver, and had great differences between males and females rats.     

Effects of n-butylparaben on steroidogenesis and spermatogenesis through changed E2 levels in male rat offspring

Parabens are widely used as antibacterial agents, which are concerned recently in the relationship between the use of parabens and reproductive toxicity. So that reassessment of the risk of parabens is needed. In this study, one of parabens, n-butylparaben (n-BP) was orally administered to pregnant Wistar rats (0, 64, 160, 400 and 1000 mg/kg/day) from gestation day (GD) 7 through postnatal day (PND) 21. Reduced anogenital distance (AGD) and delayed preputial separation (PPS) were observed in the male offspring. The weights of the testes were significantly reduced at PND 21–90. The weights of the epididymides were significantly reduced at all monitoring points, except PND 35. Seminal vesicle weights were significantly reduced on PND 21. Serum testosterone (T) was significantly decreased, especially on PND 49. The levels of 17β-estradiol (E₂) showed an increase at each of the tested points except on PND 180. Serum luteinising hormone (LH) and follicle-stimulating hormone (FSH) levels in the n-BP treated groups were lower on PND 21, 35 and 49 but elevated on PND 90 compared to control levels. n-BP reduced epididymal cauda sperm counts and daily sperm production in a dose-dependent manner; this difference was statistically significant at exposure groups of 400 and 1000 mg/kg/day. The present study strongly suggests that exposure to n-BP in utero and during lactation has adverse effects on the reproductive system in male offspring, with a no observed adverse effect level (NOAEL) of 160 mg/kg/day. To our knowledge, this is the first study that reports increased E₂ levels of male rats following n-BP exposure; we suggest that E₂ levels may be considered as biomarkers for some endocrine disrupting chemicals (EDCs).     

Dioscin,a natural steroid saponin,induces apoptosis and DNA damage through reactive oxygen species: A potential new drug for treatment of glioblastoma multiforme

Dioscin, a natural product obtained from medicinal plants shows lipid-lowering, anti-cancer and hepatoprotective effects. However, the effect of it on glioblastoma is unclear. In this study, dioscin significantly inhibited proliferation of C6 glioma cells and caused reactive oxygen species (ROS) generation and Ca²⁺ release. ROS accumulation affected levels of malondialdehyde, nitric oxide, glutathione disulfide and glutathione, and caused cell apoptosis. In addition, ROS generation caused mitochondrial damage including structural changes, increased mitochondrial permeability transition and decreased mitochondria membrane potential, which led to the release of cytochrome C, nuclear translation of programmed cell death-5 and increased activities of caspase-3,9. Simultaneously, dioscin down-regulated protein expression of Bcl-2, Bcl-xl, up-regulated expression of Bak, Bax, Bid and cleaved poly (ADP-ribose) polymerase. Also, oxygen stress induced S-phase arrest of cancer cells by way of regulating expression of DNA Topo I, p53, CDK2 and Cyclin A and caused DNA damage. In a rat allograft model, dioscin significantly inhibited tumor size and extended the life cycle of the rats. In conclusion, dioscin shows noteworthy anti-cancer activity on glioblastoma cells by promoting ROS accumulation, inducing DNA damage and activating mitochondrial signal pathways. Ultimately, we believe dioscin has promise as a new therapy for the treatment of glioblastoma.     

The relationship between DNA adduct formation by benzo[a]pyrene and expression of its activation enzyme cytochrome P450 1A1 in rat

Benzo[a]pyrene (BaP) is a human carcinogen requiring metabolic activation prior to reaction with DNA. Cytochrome P450 (CYP) 1A1 is the most important hepatic and intestinal enzyme in both BaP activation and detoxification. CYP1A2 is also capable of oxidizing BaP, but to a lesser extent. The induction of CYP1A1/2 by BaP and/or β-naphthoflavone in liver and small intestine of rats was investigated. Both BaP and β-naphthoflavone induced CYP1A expression and increased enzyme activities in both organs. Moreover, the induction of CYP1A enzyme activities resulted in an increase in formation of BaP–DNA adducts detected by ³²P-postlabeling in rat liver and in the distal part of small intestine in vivo. The increases in CYP1A enzyme activity were also associated with bioactivation of BaP and elevated BaP–DNA adduct levels in ex vivo incubations of microsomes of both organs with DNA and BaP. These findings indicate a stimulating effect of both compounds on BaP-induced carcinogenesis.     

Inhibition of methyleugenol bioactivation by the herb-based constituent nevadensin and prediction of possible in vivo consequences using physiologically based kinetic modeling

Methyleugenol (ME) occurs naturally in a variety of spices, herbs, including basil, and their essential oils. ME induces hepatomas in rodent bioassays following its conversion to a DNA reactive metabolite. In the present study, the basil constituent nevadensin was shown to be able to inhibit SULT-mediated DNA adduct formation in HepG2 cells exposed to the proximate carcinogen 1′-hydroxymethyleugenol in the presence of nevadensin. To investigate possible in vivo implications of SULT inhibition by nevadensin on ME bioactivation, the rat physiologically based kinetic (PBK) model developed in our previous work to describe the dose-dependent bioactivation and detoxification of ME in male rat was combined with the recently developed PBK model describing the dose-dependent kinetics of nevadensin in male rat. The resulting binary ME–nevadensin PBK model was used to predict the possible nevadensin mediated reduction in ME DNA adduct formation and resulting carcinogenicity at the doses of ME used by the NTP carcinogenicity study. Using these data an updated risk assessment using the Margin of Exposure (MOE) approach was performed. The results obtained point at a potential reduction of the cancer risk when rodents are orally exposed to ME within a relevant food matrix containing SULT inhibitors compared to exposure to pure ME.     

Safety of dietary conjugated α-linolenic acid (CLNA) in a neonatal pig model

The aim of the present study was to perform a short-term safety evaluation of dietary mono-conjugated α-linolenic acid isomers (CLNA; c9-t11-c15-18:3 + c9-t13-c15-18:3) using a neonatal pig model. CLNA diet was compared with three other dietary fats: (1) conjugated linoleic acid (CLA; c9-t11-18:2 + t10-c12-18:2), (2) non-conjugated n-3 PUFA and (3) n-6 PUFA. Thirty-two piglets weaned at 3 weeks of age were distributed into four dietary groups. Diets were isoenergetic and food intake was controlled by a gastric tube. Mono-CLNA diet did not significantly change body or organ weight, carcass composition and most biochemical parameters including; glucose, cholesterol, triglycerides, creatinine, blood urea nitrogen, hepatic enzymes and electrolytes levels in blood (P ⩾ 0.09). Conversely, the n-3 PUFA composition of the brain, liver and heart decreased by 6–21% in the CLNA-fed group compared to animals fed nonconjugated n-3 PUFA (P < 0.01). Responses to dietary treatments were tissue-specific, with the liver and the brain being the most deprived in n-3 PUFA. Our results support that short-term intake of mono-CLNA is safe in neonatal pigs but n-3 PUFA reduction in tissues deserves to be further investigated before using long-term nutritional supplementation in pigs and other animal models and before moving to clinical trials.     

The recovery of bladder epithelial hyperplasia caused by a melamine diet-induced bladder calculus in mice

Applying a model of bladder epithelial hyperplasia (BEH) caused by melamine-induced bladder calculus (BC), the recovery of BEH after melamine withdrawal was investigated. One experiment, comprising untreated, melamine and recovery groups, was conducted in Balb/c mice. Each group included 4 subgroups. Mice were fed normal-diet in untreated or a melamine-diet in other groups. The melamine-diet was then substituted with normal-diet in recovery group. Both of BC and BEH were observed after 14 and 56 days of melamine-diet. The BC is relatively uniform at the same melamine-diet durations. The BEH was diffuse with many mitotic figures, 4–7 rows of nuclei, and well-defined umbrella/intermediate cells. No marked differences in BEH degree were observed in the two different melamine-diet durations. On 4–42 days after melamine withdrawal, BC was not found, as the progressive regression with complete regression of BEH was observed, along with well-defined ageing/apoptotic cells in the superficial regions of BEH regression tissue. Conclusion, the melamine-induced BEH is relatively uniform, may be self-limiting in rows of nuclei, and can return to normal. Melamine withdrawal duration is critical for the BEH regression. Tissue of the BEH and its regression is ideal for exploring the renewal as well as growth biology of mammalian urothelium.     

Targeting autophagy using small-molecule compounds to improve potential therapy of Parkinson's disease

Parkinson's disease (PD), known as one of the most universal neurodegenerative diseases, is a serious threat to the health of the elderly. The current treatment has been demonstrated to relieve symptoms, and the discovery of new small-molecule compounds has been regarded as a promising strategy. Of note, the homeostasis of the autolysosome pathway (ALP) is closely associated with PD, and impaired autophagy may cause the death of neurons and thereby accelerating the progress of PD. Thus, pharmacological targeting autophagy with small-molecule compounds has been drawn a rising attention so far. In this review, we focus on summarizing several autophagy-associated targets, such as AMPK, mTORC1, ULK1, IMPase, LRRK2, beclin-1, TFEB, GCase, ERRα, C-Abelson, and as well as their relevant small-molecule compounds in PD models, which will shed light on a clue on exploiting more potential targeted small-molecule drugs tracking PD treatment in the near future.     

Immunomodulatory and cytotoxic effects of Nigella sativa and thymoquinone on rat splenocytes

Three different concentrations of Nigella sativa (N. sativa) ethanolic extract, thymoquinone (TQ), dexamethasone, and saline were examined to see whether they had any effects on cell viability, proliferation, and interleukin 4 (IL-4) and interferon-γ (IFN-γ) secretion in non-stimulated, phytohemagglutinin (PHA) and concavaline A (Con A)-stimulated splenocytes. In PHA and Con A-stimulated splenocytes, cell viability and proliferation were increased and Con A shifted cytokine profile towards Th2 balance. Dexamethasone treatment showed a suppression in viability, IFNγ and IL-4 secretion in non-stimulated and stimulated splenocytes. Extract and TQ reduced the viability and inhibited the proliferation of stimulated and non-stimulated splenocytes concentration-dependently. Higher concentrations of N. sativa (1000 mg/ml) and TQ (5 and 10 mg/ml) reduced the secretion of IL-4 in stimulated cells. Two higher concentrations of N. sativa had decreased IFNγ secretion in both stimulated and non-stimulated cells. In non-stimulated cells, only the highest and in Con A-stimulated cells, all TQ concentrations had inhibited IFNγ secretion. The highest concentration of N. sativa increased IFNγ/IL-4 ratio in both stimulated and non-stimulated cells while higher concentrations of TQ only had the same effect on stimulated cells. N. sativa and TQ showed cytotoxic inhibitory effect on rat splenocytes and on Th1/Th2 cytokines concentration-dependently. Higher concentrations of extract and TQ increased cytokines balance in Th1/Th2.     

Pro-oxidative toxicity of cells in suspension does not point to a cell cultural artefact as an explanation of the increased susceptibility of alveolar epithelial-like cells after glucocorticoid pretreatment

The influence of cell numbers on peroxide-(tertiary butylhydroperoxide (tBHP) or hydrogen peroxide-(HP)) or zinc-(zinc chloride) induced oxidative stress was assessed in alveolar epithelial-like cell lines in this work. Differences in cell numbers change the cellular glutathione and glutathione reductase activity as well as the amount of exported glutathione and therefore might influence susceptibility against oxidative stress.Toxicity due to zinc decreased, toxicity due to HP increased, while tBHP-mediated toxicity was unchanged in our experiments when cells were exposed in suspension as compared to monolayers. Toxicity of HP correlated to the glutathione content in monolayers and in cell suspensions, while zinc- or tBHP-mediated toxicity did not correlate towards glutathione.Decreasing cellular glutathione and the activity of some antioxidative enzymes by glucocorticoid pretreatment had no effect on toxicity of zinc or tBHP in L2 cells in suspensions, while toxicity in monolayers was increased. Glucocorticoid pretreatment seems to increase toxicity of HP in A549 monolayers according to the lowered protein content, while toxicity might be changed by a different way when cells are incubated as cell suspensions. No explanation as a cell culture artificial effect was observed, therefore we assume the increased toxicity after glucocorticoid pretreatment occurs in vivo as well.     

Safety evaluation of oleic-rich triglyceride oil produced by a heterotrophic microalgal fermentation process

Numbers of macro- and microalgae have been used as food sources in various cultures for centuries. Several microalgae are currently being developed as modern food ingredients. The dietary safety of oleic-rich microalgal oil produced using a heterotrophic fermentation process was assessed in a 13-week feeding trial in rats with genotoxic potential evaluated using in vitro and in vivo assays. In the genotoxicity assays, the test oil was not mutagenic in Salmonella typhimurium or Escherichia coli tester strains (⩽5000 μg/plate) with or without metabolic activation. Further, no clastogenic response occurred in chromosome aberration assays in the bone marrow of mice administered a single intraperitoneal dose (2000 mg/kg). In the subchronic study, rats consumed feed containing 0, 25,000, 50,000 or 100,000 ppm oleic-rich oil for 90 days. No treatment-related mortalities or adverse effects occurred in general condition, body weight, food consumption, ophthalmology, urinalysis, hematology, clinical chemistry, gross pathology, organ weights or histopathology. Although several endpoints exhibited statistically significant effects, none were dose-related or considered adverse. Taking all studies into consideration, the NOAEL for the oleic-rich oil was 100,000 ppm, the highest concentration tested and equivalent to dietary NOAELs of 5200 mg/kg bw/day and 6419 mg/kg bw/day in male and female rats, respectively.     

Replication priority of hepatitis C virus genotype 2a in a Chinese cohort

HCV genotypes have been documented in clinical practice. The aim of this study was to determine the replication priority of different HCV genotypes in a Chinese HCV positive cohort. Serum samples from 491 apparently healthy Chinese blood donors testing positive for HCV antibodies and naive to antiviral drug therapy were tested. Genotyping analysis showed that genotypes 1b and 2a were predominant and accounted for 77.6% of the HCV infections. Among the genotype groups, individuals infected with genotype 2a had an HCV RNA viral load (10⁸ copies/mL) about 200-fold (lg, 2.3) greater than those infected with other genotypes (10⁴–10⁵ copies/mL) indicating a replication priority of genotype 2a. However, there was no correlation between HCV genotype and antibody response suggesting that the amplification advantage of genotype 2a results from a favorable interaction with the host cellular environment. In conclusion, HCV genotypes 1b and 2a are the predominant genotypes in China and genotype 2a possesses a significant replication priority compared with the other genotypes. This suggests the existence of host cellular factors that may act as drug-targets for entirely clearing HCV infection in the future.Abbreviations: EDTA, ethylenediaminetetraacetic acid; GPT, glutamate-pyruvate transaminase; HCV, hepatitis C virus; NS3, NS4 and NS5, non-structure protein 3, 4 and 5; RdRp, RNA dependent RNA polymerase; SVR, sustained virological response     

The genetics of age-related macular degeneration (AMD) – Novel targets for designing treatment options?

Age-related macular degeneration (AMD) is a progressive disease of the central retina and the main cause of legal blindness in industrialized countries. Risk to develop the disease is conferred by both individual as well as genetic factors with the latter being increasingly deciphered over the last decade. Therapeutically, striking advances have been made for the treatment of the neovascular form of late stage AMD while for the late stage atrophic form of the disease, which accounts for almost half of the visually impaired, there is currently no effective therapy on the market. This review highlights our current knowledge on the genetic architecture of early and late stage AMD and explores its potential for the discovery of novel, target-guided treatment options. We reflect on current clinical and experimental therapies for all forms of AMD and specifically note a persisting lack of efficacy for treatment in atrophic AMD. We further explore the current insight in AMD-associated genes and pathways and critically question whether this knowledge is suited to design novel treatment options. Specifically, we point out that known genetic factors associated with AMD govern the risk to develop disease and thus may not play a role in its severity or progression. Treatments based on such knowledge appear appropriate rather for prevention than treatment of manifest disease. As a consequence, future research in AMD needs to be greatly focused on approaches relevant to the patients and their medical needs.     

The antiviral effect of jiadifenoic acids C against coxsackievirus B3

Coxsackievirus B type 3 (CVB3) is one of the major causative pathogens associated with viral meningitis and myocarditis, which are widespread in the human population and especially prevalent in neonates and children. These infections can result in dilated cardiomyopathy (DCM) and other severe clinical complications. There are no vaccines or drugs approved for the prevention or therapy of CVB3-induced diseases. During screening for anti-CVB3 candidates in our previous studies, we found that jiadifenoic acids C exhibited strong antiviral activities against CVB3 as well as other strains of Coxsackie B viruses (CVBs). The present studies were carried out to evaluate the antiviral activities of jiadifenoic acids C. Results showed that jiadifenoic acids C could reduce CVB3 RNA and proteins synthesis in a dose-dependent manner. Jiadifenoic acids C also had a similar antiviral effect on the pleconaril-resistant variant of CVB3. We further examined the impact of jiadifenoic acids C on the synthesis of viral structural and non-structural proteins, finding that jiadifenoic acids C could reduce VP1 and 3D protein production. A time-course study with Vero cells showed that jiadifenoic acids C displayed significant antiviral activities at 0–6 h after CVB3 inoculation, indicating that jiadifenoic acids C functioned at an early step of CVB3 replication. However, jiadifenoic acids C had no prophylactic effect against CVB3. Taken together, we show that jiadifenoic acids C exhibit strong antiviral activities against all strains of CVB, including the pleconaril-resistant variant. Our study could provide a significant lead for anti-CVB3 drug development.KEY WORDS: CVB3, Jiadifenoic acids C, Antiviral activityAbbreviations: CAR, coxsackievirus and adenovirus receptor; CPE, cytopathic effect; CVB3, coxsackievirus B type 3; CVBs, coxsackie B viruses; DAF, decay accelerating factor; DCM, dilated cardiomyopathy; IC₅₀, 50% inhibitory concentration; IRES, internal ribosomal entry site; MOI, multiplicity of infection; NTR, non-translated region; RBV, ribavirin; RdRp, RNA-dependent RNA polymerase; SI, selectivity index; Vero, African green monkey kidney cells