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.     

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.     

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.     

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).     

Effect of prenatal manganese intoxication on [3H]glucose uptake in the brain of rats lesioned as neonates with 6-hydroxydopamine

In the present study we examined the effects of prenatal manganese (Mn) intoxication on [³H]glucose uptake in the brain of rats lesioned as neonates with 6-hydroxydopamine (6-OHDA). MnCl₂ ? 4H2O (10,000 ppm) was added to the drinking water of pregnant Wistar rats for the duration of pregnancy. On the day of parturition, Mn was discontinued as an additive to the drinking water. The control group consisted of rats that consumed water without Mn. Three days after birth, rats in both groups (control and Mn) were pretreated with desipramine hydrochloride (20 mg/kg) and pargyline hydrochloride (50 mg/kg) and injected bilaterally icv with one of three doses of 6-OHDAhydrobromide (15 μg, 30 μg or 67 μg base form in saline on each side) or with saline (control). 6-[³H]-Dglucose (500 μCi/kg, ip) was administered to male offspring in adulthood; after 15 min, brain specimens were taken (frontal cortex, hippocampus, striatum, thalamus with hypothalamus, pons and cerebellum) for determination of radioactivity in a liquid scintillation counter. Low dose 6-OHDA (15 μg icv) increased [³H]glucose uptake in all brain regions (p < 0.05) in both control and Mnintoxicated animals. In rats lesioned with a moderate dose of 6-OHDA(30 μg icv), [3H]glucose uptake was unaltered in both control and Mn-exposed rats. High dose 6-OHDA(67 μg icv) reduced [³H]glucose uptake in all brain regions of Mn-exposed rats (except for cerebellum) compared with the saline group (all, p < 0.05). There was no change in regional brain uptake of [³H]glucose in control rats. In conclusion, this study shows that mild neuronal insult (15 μg icv 6-OHDA) increased glucose uptake in the brain while severe damage (concomitant 60 μg icv 6-OHDA and Mn treatment) significantly diminished this process.     

Transient activation of protein kinase C contributes to fluoride-induced apoptosis of rat erythrocytes

Role of PKC in fluoride-induced apoptosis of rat erythrocytes was studied in vitro and in vivo. Treatment of erythrocytes with 5 mM NaF for 1–24 h caused progressive accumulation of cytosolic Ca²⁺ and PS exposure at outer membrane surface. After 1 h, these processes were suppressed by PKC inhibitors staurosporine, GF 109203X and chelerythrine, but increased by PKC activator PMA. Following 24 h, NaF-induced Ca²⁺ uptake and PS externalization were partly prevented by PMA or staurosporine, but not by GF 109293X and chelerythrine. Application of PP inhibitor OA augmented NaF-induced cell responses within 1 h, but not after 24 h. Incubation of erythrocytes with 0.1–10 mM NaF for 1 h produced a dose-dependent PKCα translocation from cytosol to membranes with appearance of active PKM fragment. 24 h NaF exposure led to complete loss of cytosolic PKCα and proteolysis of membrane PKCα. Besides, NaF weakly stimulated membrane PKCζ, although its subcellular distribution was not altered. Thus, transient PKCα activation/translocation positively contributes to NaF-induced apoptosis in vitro. Consumption of 2–20 ppm fluoride by the rats for 12 months also induced dose-dependent PKCα translocation to membranes and activation of membrane PKCζ, what indicates that PKC stimulation is an important physiological mechanism of fluoride toxicity.     

The effect of phytosterol protects rats against 4-nitrophenol-induced liver damage

We investigated the effect of phytosterol (PS) in regard to liver damage induced by 4-nitrophenol (PNP). Twenty rats were randomly divided into four groups (Control, PS, PNP, and PNP + PS). The PS and PNP + PS groups were pretreated with PS for one week. The PNP and PNP + PS groups were injected subcutaneously with PNP for 28 days. The control group received a basal diet and was injected with vehicle alone. Treatment with PS prevented the elevation of the total bilirubin levels, as well as an increase in serum alkaline transaminase and aspartate transaminase, which are typically caused by PNP-induced liver damage. Histopathologically showed that liver damage was significantly mitigated by PS treatment. However, there was no significant change in antioxidant enzyme activities, and the Nrf2-antioxidant system was not activated after treatment with PS. These results suggest that PS could mitigate liver damage induced by PNP, but does not enhance antioxidant capacity.     

Supersaturated polymeric micelles for oral cyclosporine A delivery

Polymeric micelles provide a promising platform for improving oral absorption of poorly soluble drugs. However, improved understanding of how drug retention within the hydrophobic micelle core can reduce drug absorption is required. We designed supersaturated polymeric micelles (Super-PMs) to increase molecularly dissolved drug concentration and gain an insight into the effect of the degree of supersaturation on oral absorption of cyclosporine A (CsA) in rats. The drug release from Super-PMs increased with an increase in initial supersaturation degrees in micelles. The cellular uptake of coumarin-6 was reduced by the retention of drug in polymer micelles. The transport flux of CsA across Caco-2 monolayer was increased with initial supersaturation degrees of 0.81–3.53 (p < 0.05). However, increase in supersaturation to 5.64 actually resulted in decreased CsA transport. The same trend was observed in a rat in vivo absorption study, in which the highest bioavailability of 134.6 ± 24.7% (relative to a commercial product, Sandimmun Neoral®, p < 0.01) was achieved when the supersaturation degree was 3.53. These results demonstrated that Super-PMs were a promising drug delivery system for compounds with low aqueous solubility. This study also provided an experimental proof for the hypothesis that moderately supersaturated formulations are valuable alternative to high supersaturation formulations, resulting in optimal in vivo performance, and the degree of supersaturation should be carefully controlled to optimize drug absorption.     

Acute and subchronic toxicity studies of seabuckthorn (Hippophae rhamnoides L.) oil in rodents

Seabuckthorn (Hippophae rhamnoides L.) has been traditionally used as medicine and nutritional supplement for a long period of time. However, information on the systemic toxicity and safety evaluation of seabuckthorn and its extracts is still scarce. The purpose of this study was to evaluate the potential toxicity of seabuckthorn oil by an acute oral toxicity study in mice and a 90-day repeated oral toxicity study in rats. No mortality or signs of toxicity was observed in mice treated with 20 mL/kg body weight seabuckthorn oil in the acute toxicity study. In the subchronic toxicity study, 80 Sprague-Dawley rats (10 animals per sex per treatment group) were administrated with 10, 5, 2.5 and 0 (control) mL/kg body weight of seabuckthorn oil daily for 90 days by gavage. There were no signs of toxicity and treatment-related changes in rats treated with seabuckthorn oil on mortality, body and organ weights, food consumption, blood biochemistry and hematology, gross necropsy and histopathological examinations. Based on the finding of this study, the maximum tolerated dose of seabuckthorn oil was >20 mL/kg for mice for acute toxicity study, and the no-observed-adverse-effect level was 10 mL/kg body weight for both male and female rats for 90-day toxicity study.     

The Fusarium toxin zearalenone and deoxynivalenol affect murine splenic antioxidant functions,interferon levels,and T-cell subsets

This study aimed to evaluate the effects of the Fusarium toxin zearalenone (ZEA) and deoxynivalenol (DON) on splenic antioxidant functions, IFN levels, and T-cell subsets in mice. Herein, 360 mice were assigned to nine groups for a 12-day study. Mice were administered an intraperitoneal injection for 4 consecutive days with different concentrations of ZEA alone, DON alone, or ZEA + DON. Spleen and blood samples were collected on days 0, 3, 5, 8, and 12. Mice in each of the experimental groups showed dysreglated splenic antioxidant functions, IFN levels, and T-cell subset frequencies, suggesting that the immune system had been affected. The ZEA + DON-treated groups, especially the group that received a higher concentration of ZEA + DON (Group D2Z2), showed more obvious effects on the dysregulation of splenic antioxidant functions, IFN levels, and T-cell subsets. This finding suggested that DON and ZEA exerted synergistic effects.     

A BALB/c mouse model for assessing the potential allergenicity of proteins: Comparison of allergen dose,sensitization frequency,timepoint and sex

The present study was to investigate the possibility of using the BALB/c mouse as an animal model for assessing the potential allergenicity of proteins.Specific IgE and IgG1 against ovalbumin were induced by dosing BALB/c mice via intraperitoneal injection (absence of adjuvant). The effects of various allergen doses (5 mg, 0.5 mg or 0.05 mg OVA), sensitization times (twice or five times), timepoints (day 14 or day 28) and sex (male or female) were studied. IL-4, IFN-γ, OVA-specific IgE and IgG1 were measured by enzyme-linked immunosorbent assay (ELISA).A general finding was that mice treated with 0.05 mg OVA had the highest OVA-specific IgE and IgG1, statistically significant higher specific IgE and IgG1 were observed in groups sensitized five times than twice, OVA-specific IgE and IgG1 on day 28 were statistically higher than day 14, and higher IL-4 was observed in OVA-allergic mice than control mice.These results demonstrate that the BALB/c mouse model treated with 0.05 mg OVA intraperitoneally on days 0, 3, 6, 9, 12 might be used for further experiments. OVA-specific IgE and IgG1 should be detected on day 28. Further studies including reproducibility and other conditions were required before using the BALB/c mouse model for assessing the potential allergenicity of proteins.     

Effects of endocrine disrupting chemicals on in vitro global DNA methylation and adipocyte differentiation

Recent studies suggest that endocrine disrupting chemicals (EDCs) may form a risk factor for obesity by altering energy metabolism through epigenetic gene regulation. The goal of this study is to investigate the effects of a range of EDCs with putative obesogenic properties on global DNA methylation and adipocyte differentiation in vitro. Murine N2A and human SK-N-AS neuroblastoma cells and murine preadipocyte fibroblasts (3T3-L1) were exposed to tributyltin (TBT), diethylstilbestrol (DES), bisphenol A (BPA), 2,3,7,8-tetrachlorodibenzo-[p]-dioxin (TCDD), 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB-153), hexachlorobenzene (HCB), hexabromocyclododecane (HBCD), 2,2′,4,4′-tetrabrominated diphenyl ether (BDE-47) , perfluorinated octyl acid (PFOA) and perfluorinated octyl sulfonate (PFOS). A modest decrease in global DNA methylation was observed in N2A cells exposed to 10 μM DES, BPA, TCDD, BDE-47, PCB-153 and 1 μM HCB, but no changes were found in the human SK-N-AS cells. We reveal for the first time that BDE-47 increases adipocyte differentiation in a dose-dependent manner (2.5–25 μM). Adipocyte differentiation was also enhanced by TBT (?10 nM) and BPA (>10 μM) and inhibited by TCDD (?0.1 nM). The other chemicals showed either modest or no effects on adipocyte differentiation at the concentrations tested (PFOA, PFOS and HBCD at 10 μM; PCB-153, 3.4 μM and HCB, 1 μM). This study demonstrates that selected EDCs can induce functional changes in murine adipocyte differentiation in vitro which are accompanied by decreased global DNA methylation.     

4-Week repeated dose oral toxicity study of N-ethyl-2-pyrrolidone in Sprague Dawley rats

The solvent N-ethyl-2-pyrrolidone (NEP) was evaluated in a 4-week repeated dose study in rats. NEP diluted in distilled water was orally administered by gavage to male and female Sprague-Dawley rats at doses of 0 (vehicle control), 5, 50, and 250 mg/kg/day for 28 consecutive days. Transient decreases in the body weight and in the body weight gain of the males was observed during the first days of treatment at the 50 and 250 mg/kg/day doses. There was a marked increase in urine volume at the beginning of treatment in males and female rats at doses of 50 and 250 mg/kg/day. No biologically significant differences were observed in hematological and clinical chemistry values in males and females at necropsy. Histological examination revealed an increase in hyaline droplets in the renal tubules of the kidneys and hepatocellular centrilobular hypertrophy in the liver of males at 250 mg/kg/day. Cytochrome P450 concentration in liver microsomes was slightly increased at 250 mg/kg/day in males. The results of this study demonstrate that NEP has mild to no effects at doses up to 250 mg/kg/day when administered orally to rats for 28 days with males being more susceptible than females.     

A comprehensive study on in vitro and in vivo toxicological evaluation of Artemisia capillaris

Artemisia capillaris (AC) has been used as an alternative therapy in obesity, atopic dermatitis, and liver diseases through several biological activity including anti-steatotic, antioxidant, and anti-inflammatory activities. Despite its ethnomedicinal benefits, no sufficient background information is available about the long-term safety and genotoxicity of the AC extract. Therefore, the present study was carried out to investigate the 13-week subchronic toxicity and genotoxicity of the AC extract according to the test guidelines published by the Organization for Economic Cooperation and Development. In the 13-week toxicity study using doses of 25, 74, 222, 667, and 2000 mg/kg body weight, oral administration of the AC extract in male and female rats did not result in any significant adverse effects in food/water consumption, body weight, mortality, hematology, serum biochemistry, organ weight and histopathology. Accordingly, the no-observed-adverse-effect level in rats of both genders was established for the AC extract at 2000 mg/kg/day, the highest dose level tested. In addition, the AC extract was not genotoxic in a battery of tests including Ames test, in vitro chromosome aberration assay and in vivo micronucleus assay. In conclusion, we demonstrated that the AC extract is considered as a safe traditional medicine for human consumption.     

Protective effects of timosaponin B-II on high glucose-induced apoptosis in human umbilical vein endothelial cells

This study was designed to investigate the action of timosaponin B-II, a main bioactive compound in Anemarrhena asphodeloides Bunge, on the prevention from high glucose-induced cytotoxicity and apoptosis in human umbilical vein endothelial cells (HUVECs) and the potential mechanisms involved. The results showed that compared with the normal control group, exposure of HUVECs to high glucose media for 72 h resulted in a significant increase in lactates dehydrogenise release, reactive oxygen species production, Caspase-3 activity and the percentage of apoptotic cells (p < 0.01). However, pretreatment with timosaponin B-II significantly increased the viability of HUVECs and decreased lactates dehydrogenise release, Caspase-3 activity and the apoptosis rate in a concentration-dependent manner (p < 0.05). In addition, timosaponin B-II notably decreased the amount of reactive oxygen species and malondialdehyde, as well as promoted glutathione peroxidase activity, endothelial nitric oxide synthase activity and nitric oxide release (p < 0.05). These results suggest that timosaponin B-II has the antiapoptotic effect in endothelial cells through inhibition of high glucose-induced oxidative stress and has the potential for preventing diabetic cardiovascular complications.     

T cell--associated immunoregulation and antiviral effect of oxymatrine in hydrodynamic injection HBV mouse model

Although oxymatrine (OMT) has been shown to directly inhibit the replication of hepatitis B virus (HBV) in vitro, limited research has been done with this drug in vivo. In the present study, the antiviral effect of OMT was investigated in an immunocompetent mouse model of chronic HBV infection. The infection was achieved by tail vein injection of a large volume of DNA solution. OMT (2.2, 6.7 and 20 mg/kg) was administered by daily intraperitoneal injection for 6 weeks. The efficacy of OMT was evaluated by the levels of HBV DNA, hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) and hepatitis B core antigen (HBcAg). The immunoregulatory activity of OMT was evaluated by serum ELISA and flow cytometry. Results shows that OMT at 20 mg/kg inhibited HBV replication, and it was more efficient than entecavir (ETV) in the elimination of serum HBsAg and intrahepatic HBcAg. In addition, OMT accelerated the production of interferon-γ (IFN-γ) in a dose-dependent manner in CD4⁺ T cells. Our findings demonstrate the beneficial effects of OMT on the enhancement of immunological function and in the control of HBV antigens. The findings suggest this drug to be a good antiviral therapeutic candidate for the treatment of HBV infection.     

Modulatory effects of sesamol in dinitrochlorobenzene-induced inflammatory bowel disorder in albino rats

BackgroundInflammatory bowel disease (IBD) is a chronic inflammatory condition of gastrointestinal tract of immune, genetic and environmental origin. In the present study, we examined the effect of sesamol (SES), the main anti-oxidative constituent of Sesamum indicum (sesame seed) Linn. in the dinitrochlorobenzene (DNCB)-induced model for IBD in rats.MethodsThe groups were divided into normal control, DNCB control, SES and sulfasalazine (SS). On day 24, the rats were killed, colon removed and the macroscopic, biochemical and histopathological evaluations were performed.ResultsThe levels of MPO, TBARS and nitrite increased significantly (p < 0.05) in the DNCB group, whereas reduced significantly in the SES, SS treated groups. Serum nitrite levels were found to be insignificant between the different groups. IL-6 and TNF-α levels were significantly high in the DNCB group.ConclusionsWe conclude the mucosal protective effect of SES on colon due to its potent antioxidant actions. Further investigation is required in a chronic model of different rodent strain for its role involved in the cytokine pathway.     

Intake of anthocyanidins pelargonidin and cyanidin reduces genotoxic stress in mice induced by diepoxybutane,urethane and endogenous nitrosation

Pelargonidin (PEL) and cyanidin (CYN) are among the six most abundant anthocyanidins which provide red, blue and purple colors to fruits and vegetables. Health benefits associated with intake of anthocyanins have been attributed mainly to antioxidant activity of these color pigments. The aim of our present study was to assess in mice the impact of PEL and CYN intake on genotoxic stress induced by DNA damaging environmental toxicants. These anthocyanidins were administered by gavage to mice before exposure to genotoxic carcinogens diepoxybutane (DEB) and urethane (URE). In addition, the inhibitory effect of PEL and CYN on endogenous nitrosation was evaluated by using a model nitrosation reaction mixture consisting of methyl urea (MU) + sodium nitrite (SN) which reacts in the stomach to form the carcinogenic methyl nitrosourea (MNU). All the test doses of PEL (2.5–20 mg/kg) and CYN (1–4 mg/kg) significantly reduced the genotoxicity of DEB. A dose-related increase was observed for antigenotoxicity of PEL against URE. The lowest test-dose of CYN showed maximum protection against URE. Co-administration of PEL/CYN with the nitrosation reaction mixture led to reduction in genotoxicity. CYN was more effective as an inhibitor of endogenous nitrosation. Combination of PEL with ascorbic acid (AA) enhanced the antinitrosating effect when compared to that with each phytochemical alone. The results of our present study indicate that common anthocyanidins PEL and CYN can play a major role in reducing genotoxic stress induced by environmental toxicants.