Ptaquiloside from bracken (Pteridium spp.) inhibits tumour-infiltrating CD8+ T cells in HPV-16 transgenic mice

Bracken is a fern with worldwide distribution. Exposure to bracken toxins such as ptaquiloside is hypothesized to increase the risk of papillomavirus-related cancers of the upper digestive tract. Ptaquiloside is thought to be an immunosupressor, thus allowing for the development of viral lesions. We have used a human papillomavirus type 16-transgenic (K14-HPV16) mouse model to study the effects of ptaquiloside on tumour-infiltrating CD8⁺ T lymphocytes, which are critical players in anti-tumour immunity. HPV16^+/− mice received ptaquiloside (0.5 mg/mouse/week) for 10 weeks. These were then euthanized at 30 weeks of age, along with age-matched untreated controls. Skin samples were enzymatically digested and CD8⁺ T cells analysed for CD107a and CD44 surface expression. Ptaquiloside-exposed HPV16^+/− mice showed a significantly decreased percentage (P < 0.05) of CD8⁺CD107a⁺ and CD8⁺CD44 ⁺ T cells when compared with untreated HPV16^+/− animals. Histologically, 100% of ptaquilosidetreated mice showed diffuse epidermal dysplasia, compared with 50% of the untreated mice. These findings suggest that ptaquiloside exerts an immunosuppressive role by decreasing CD8⁺ T cell activation and degranulation in HPV-induced lesions. Given the key role of CD8⁺ T lymphocytes against HPV-induced lesions, this effect is likely to contribute for viral persistence, tumour progression and increased aggressiveness in patients with HPV-related malignancies.     

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.     

Fish cell lines as a tool for the ecotoxicity assessment and ranking of engineered nanomaterials

Risk assessment of engineered nanomaterials (ENMs) is being hindered by the sheer production volume of these materials. In this regard, the grouping and ranking of ENMs appears as a promising strategy. Here we sought to evaluate the usefulness of in vitro systems based on fish cell lines for ranking a set of ENMs on the basis of their cytotoxicity. We used the topminnow (Poeciliopsis lucida) liver cell line (PLHC-1) and the rainbow trout (Oncorhynchus mykiss) fibroblast-like gonadal cell line (RTG-2). ENMs were obtained from the EU Joint Research Centre repository. The size frequency distribution of ENM suspensions in cell culture media was characterized. Cytotoxicity was evaluated after 24 h of exposure. PLHC-1 cells exhibited higher sensitivity to the ENMs than RTG-2 cells. ZnO-NM was found to exert toxicity mainly by altering lysosome function and metabolic activity, while multi-walled carbon nanotubes (MWCNTs) caused plasma membrane disruption at high concentrations. The hazard ranking for toxicity (ZnO-NM > MWCNT ≥ CeO₂-NM = SiO₂-NM) was inversely related to the ranking in size detected in culture medium. Our findings reveal the suitability of fish cell lines for establishing hazard rankings of ENMs in the framework of integrated approaches to testing and assessment.     

Estimation of in vivo and in vitro exposure to bisphenol A as food contaminant

The goal of this cross-sectional study was to examine the occurrence of bisphenol A (BPA) in the morning spot urine taken from 145 female volunteers of various ages. Total urine BPA concentration was detected in 38.6% samples in the 0.92–70.96 μg/g Cr range. The majority of BPA + women belonged to the 25 + body mass index (BMI) group (54.5% were overweight and 43.4% were obese women). Occurrence of BPA in the urine samples was higher at 40 + ages. The maximum BPA concentration of 70.96 μg/g Cr was detected in the urine sample of an obese woman. It is known that BPA is highly toxic in vitro. In this study BPA impaired significantly the growth of all investigated cell lines, i.e. the EC₅₀ values were reached at very low concentrations, in the range from 3.24 to 34.85 μg/mL. The obtained in vivo results suggest that a higher exposure to BPA could contribute to weight problems in women and the absence of the BPA in vitro selective toxicity studies indicates to its general toxic mode of action and raises awareness of the health risks associated with its ubiquitous presence in the environment.     

Hepatotoxic effect of ochratoxin A and citrinin,alone and in combination,and protective effect of vitamin E: In vitro study in HepG2 cell

Ochratoxin A (OTA) and citrinin (CTN) are the most commonly co-occurring mycotoxins in a wide variety of food and feed commodities. The major target organ of these toxins is kidney but liver could also be a target organ. The combined toxicity of these two toxins in kidney cells has been studied but not in liver cell. In this study HepG2 cells were exposed to OTA and CTN, alone and in combination, with a view to compare the molecular and cellular mechanisms underlying OTA, CTN and OTA + CTN hepatotoxicity. OTA and CTN alone as well as in combination affected the viability of HepG2 cells in a dose-dependent manner. OTA + CTN, at a dose of 20% of IC₅₀ of each, produced effect almost similar to that produced by either of the toxins at its IC₅₀ concentration, indicating that the two toxins in combination act synergistically. The cytotoxicity of OTA + CTN on hepatocytes is mediated by increased level of intracellular ROS followed/accompanied by DNA strand breaks and mitochondria-mediated intrinsic apoptosis. Co-treatment of vitamin E (Vit E) with OTA, CTN and OTA + CTN reduced the levels of ROS and the cytotoxicity. But the genotoxic effect of OTA and OTA + CTN was not completely alleviated by Vit E treatment whereas the DNA damage as caused by CTN when treated alone was obviated, indicating that OTA induces DNA damage directly whereas CTN induces ROS-mediated DNA damage and OTA + CTN combination induces DNA damage not exclusively relying on but influenced by ROS generation. Taken together, these findings indicate that OTA and CTN in combination affect hepatocytes at very low concentrations and, thereby, pose a potential threat to public and animal health.     

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.     

Assessment of oxidative stress responses and the cytotoxic and genotoxic potential of the herbicide tembotrione in HepG2 cells

Tembotrione is a triketone herbicide, usually used for post-emergence weed control in corn. Currently, there is little or no published data on its genotoxicity to human cells either in vitro or in vivo. This study evaluated the impact of acute (4 and 24 h) exposure to low concentrations of tembotrione [corresponding to the acceptable daily intake (0.17 μg/mL), residential exposure level (0.002 μg/mL) and acceptable operator exposure level (0.0012 μg/mL)] on human hepatocellular carcinoma cell line HepG2, using biomarkers of oxidative stress, CCK-8 colorimetric assay for cell viability, alkaline comet assay, and cytokinesis-block micronucleus “cytome” assay. Tembotrione applied at concentrations likely to be encountered in occupational and residential exposures induced cytogenetic outcomes in non-target cells despite non-significant changes in the values of oxidative stress biomarkers. We assume that the observed effects were mainly the consequence of impaired metabolic pathways in HepG2 cells due to the inhibition of the enzyme 4-hydroxyphenyl-pyruvate-dioxygenase by tembotrione, which possibly caused a depletion of folate levels leading to excess formation of nuclear buds in the affected cells. Regardless of the fact that tembotrione was previously reported negative for mutations and chromosome aberrations in vitro, our findings call for more precaution in its use.     

Comparison of the impact of the Tobacco Heating System 2.2 and a cigarette on indoor air quality

The impact of the Tobacco Heating System 2.2 (THS 2.2) on indoor air quality was evaluated in an environmentally controlled room using ventilation conditions recommended for simulating “Office”, “Residential” and “Hospitality” environments and was compared with smoking a lit-end cigarette (Marlboro Gold) under identical experimental conditions. The concentrations of eighteen indoor air constituents (respirable suspended particles (RSP) < 2.5 μm in diameter), ultraviolet particulate matter (UVPM), fluorescent particulate matter (FPM), solanesol, 3-ethenylpyridine, nicotine, 1,3-butadiene, acrylonitrile, benzene, isoprene, toluene, acetaldehyde, acrolein, crotonaldehyde, formaldehyde, carbon monoxide, nitrogen oxide, and combined oxides of nitrogen) were measured. In simulations evaluating THS 2.2, the concentrations of most studied analytes did not exceed the background concentrations determined when non-smoking panelists were present in the environmentally controlled room under equivalent conditions. Only acetaldehyde and nicotine concentrations were increased above background concentrations in the “Office” (3.65 and 1.10 μg/m³), “Residential” (5.09 and 1.81 μg/m³) and “Hospitality” (1.40 and 0.66 μg/m³) simulations, respectively. Smoking Marlboro Gold resulted in greater increases in the concentrations of acetaldehyde (58.8, 83.8 and 33.1 μg/m³) and nicotine (34.7, 29.1 and 34.6 μg/m³) as well as all other measured indoor air constituents in the “Office”, “Residential” and “Hospitality” simulations, respectively.     

Role of TLR ligands in epicutaneously induced contrasuppression

Our previous work showed that epicutaneous (EC) immunization in mice with protein antigen (Ag) induced an Ag-independent unresponsiveness mediated by suppressor CD4⁺8⁺ T cells (Ts), which inhibited contact hypersensitivity (CS). Simultaneous EC immunization with Ag and various Toll-like receptor (TLR) ligands reversed skin-induced suppression. Our present study shows that this process activates Ag-specific T contrasuppressor (Tcs) cells and leads to the protection of CS effector T cells from suppression. Epicutaneous immunization with Ag and the TLR4 ligand lipopolysaccharide (LPS) led to a significant increase in IFN-γ production by lymph node and spleen cells. Ag and TLR ligands, like LPS, CpG or lipoteichoic acid did not need to be applied concomitantly to the skin. An identical contrasuppressive effect was observed when the Ag and TLR ligands were deposited on distant skin areas, suggesting that both the generation of Ts and Tcs are independent. To corroborate this finding, we used a model system that uses macrophages (Mf) as Ag-presenting cells. Mf labeled in vitro with Ag (Mf-Ag) induced, upon intravenous (iv) administration, an unresponsiveness reaction that was mediated by Ts cells. When treated simultaneously with LPS-treated Mf (Mf-Ag-LPS), a TLRligand could induce CS. Both the Ag and the LPS signal could be uncoupled i.e., Mf-Ag and Mf-LPS given at separate time points (with an 1 h interval between injections) induced immunity.We also found that LPS-treated Mf also produced significant amounts of IL-12, a cytokine that has well-known anti-tolerogenic properties. Our experiments suggest that reversal of EC-induced suppression by TLR-ligands may be a potential tool to increase the immunogenicity of weakly immunogenic antigens.     

Acute and subacute toxicity studies of CMICE-013, a novel iodinated rotenone-based myocardial perfusion tracer,in Sprague Dawley rats and Gottingen minipigs

PurposeExtensive acute and subacute toxicities studies are required to evaluate the toxicological profile of the novel cardiac perfusion imaging tracer ¹²³I-CMICE-013 to support applications for clinical trials.MethodsSprague-Dawley rats and Gottingen minipigs received injections of non-radioactive 127I-CMICE-013 at two dosage levels of 1 and 5 μg/kg, and vehicle buffer as control. In the acute toxicity studies, each animal was injected on two occasions 24 h apart and then underwent a 14-day recovery period; in the subacute study, animals received daily injections for 14 days continuously. The health status and mortality of test animals were monitored daily and body weight, food consumption, physiological and biochemical parameters were measured at various time points during the study. Animals were euthanized at the end of the studies and dissected for pathologic examination of organs and tissues.ResultsThe acute and subacute administrations of injections of the non-radioactive CMICE-013 in rats and minipigs were well tolerated. Little to no dosing-related adverse effects were observed in animal body and organ weights, hematology, coagulation, clinical chemistry, urinalysis, ophthalmoscopy, electrocardiograms, heart rates, blood pressure, macroscopic and microscopic examination of the preserved animal tissues including the brain.ConclusionThe lack of adverse effects from acute and subacute dosing suggest that the CMICE-013 injection solution has a reasonable safety margin within the designed concentration range to be utilized in imaging applications. The dosage level of 5 μg/kg was considered the no adverse effect level for both rats and minipigs based on our acute and subacute studies.     

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.     

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.     

Abrogation of HnRNP L enhances anti-PD-1 therapy efficacy via diminishing PD-L1 and promoting CD8+ T cell-mediated ferroptosis in castration-resistant prostate cancer

Owing to incurable castration-resistant prostate cancer (CRPC) ultimately developing after treating with androgen deprivation therapy (ADT), it is vital to devise new therapeutic strategies to treat CRPC. Treatments that target programmed cell death protein 1 (PD-1) and programmed death ligand-1 (PD-L1) have been approved for human cancers with clinical benefit. However, many patients, especially prostate cancer, fail to respond to anti-PD-1/PD-L1 treatment, so it is an urgent need to seek a support strategy for improving the traditional PD-1/PD-L1 targeting immunotherapy. In the present study, analyzing the data from our prostate cancer tissue microarray, we found that PD-L1 expression was positively correlated with the expression of heterogeneous nuclear ribonucleoprotein L (HnRNP L). Hence, we further investigated the potential role of HnRNP L on the PD-L1 expression, the sensitivity of cancer cells to T-cell killing and the synergistic effect with anti-PD-1 therapy in CRPC. Indeed, HnRNP L knockdown effectively decreased PD-L1 expression and recovered the sensitivity of cancer cells to T-cell killing in vitro and in vivo, on the contrary, HnRNP L overexpression led to the opposite effect in CRPC cells. In addition, consistent with the previous study, we revealed that ferroptosis played a critical role in T-cell-induced cancer cell death, and HnRNP L promoted the cancer immune escape partly through targeting YY1/PD-L1 axis and inhibiting ferroptosis in CRPC cells. Furthermore, HnRNP L knockdown enhanced antitumor immunity by recruiting infiltrating CD8⁺ T cells and synergized with anti-PD-1 therapy in CRPC tumors. This study provided biological evidence that HnRNP L knockdown might be a novel therapeutic agent in PD-L1/PD-1 blockade strategy that enhanced anti-tumor immune response in CRPC.     

Detection of ECG effects of (2R,4R)-monatin,a sweet flavored isomer of a component first identified in the root bark of the Sclerochitin ilicifolius plant

Enzymatically-synthesized (2R,4R)-monatin has, due to its pure sweet taste, been evaluated for potential use in foods. Non-clinical studies have shown that (2R,4R)-monatin is well tolerated at high dietary concentrations, is not genotoxic/mutagenic, carcinogenic, or overtly toxic. In a pharmacokinetic and metabolism study involving 12 healthy males, consumption of a single oral dose (2 mg/kg) of (2R,4R)-monatin resulted in a small reduction of heart rate and prolongation of the QTcF interval of 20–24 ms, corresponding to the time of peak plasma levels (t_max). These findings were evaluated in a cross-over thorough QT/QTc study with single doses of 150 mg (2R,4R)-monatin, placebo and positive control (moxifloxacin) in 56 healthy males. Peak (2R,4R)-monatin plasma concentration (1720 ± 538 ng/mL) was reached at 3.1 h (mean t_max). The placebo-corrected, change-from-baseline QTcF (ΔΔQTcF) reached 25 ms three hours after dosing, with ΔΔQTcF of 23 ms at two and four hours. Using exposure response (QTc) analysis, a significant slope of the relationship between (2R,4R)-monatin plasma levels and ΔΔQTcF was demonstrated with a predicted mean QT effect of 0.016 ms per ng/mL. While similarly high plasma levels are unlikely to be achieved by consumption of (2R,4R)-monatin in foods, QTc prolongation at this level is a significant finding.     

A dietary two-generation reproductive toxicity study of (2R,4R)-monatin salt in Crl:CD(SD) rats

(2R,4R)-Monatin salt [sodium/potassium 2R,4R-2-amino-4-carboxy-4-hydroxy-5-(3-indolyl) pentanoate] was fed at 5000, 15,000, or 35,000 ppm to Crl:CD(SD) rats over two generations. Reduced body weights were observed at all dose levels. Sustained effect on body weight gain at 35,000 ppm in the F₀ and F₁ parental animals was associated with lower feed efficiency, soft stool, and slightly lower numbers of implantation sites. Lower numbers of pups born and live litter size at 35,000 ppm were considered secondary to slightly lower numbers of former implantation sites in the dams. Spermatogenic endpoints, estrous cyclicity, reproductive performance, mean gestation length, and parturition were unaffected in the F₀ and F₁ generations. There were no effects on F₁ and F₂ generation postnatal survival. Reduced pre-weaning pup body weights at 35,000 ppm resulted in lower F₁ and F₂ body weights at study termination. Slight delays in pubertal landmarks in the F₁ offspring were considered secondary to the reduced pup body weights. The no-observed-adverse-effect level (NOAEL) was 15,000 ppm for systemic, reproductive, and neonatal effects based on test article-related effects on body weight and food efficiency, slight decrease in maternal implantation sites and corresponding reduction in live litter size, and reductions in pre-weaning pup body weights at 35,000 ppm.     

Development of Caco-2 cells co-expressing CYP3A4 and NADPH-cytochrome P450 reductase using a human artificial chromosome for the prediction of intestinal extraction ratio of CYP3A4 substrates

The Caco-2 cells co-expressing cytochrome P450 (CYP) 3A4 and NADPH-cytochrome P450 reductase (CPR) were developed using a human artificial chromosome (HAC) vector. The CYP3A4 and CPR genes were cloned into the HAC vector in CHO cells using the Cre-loxP system, and the microcell-mediated chromosome transfer technique was used to transfer the CYP3A4-CPR-HAC vector to Caco-2 cells. After seeding onto semipermeable culture inserts, the CYP3A4-CPR-HAC/Caco-2 cells were found to form tight monolayers, similar to the parental cells, as demonstrated by the high transepithelial electrical resistance (TEER) value and comparable permeability of non-CYP3A4 substrates between parent and CYP3A4-CPR-HAC/Caco-2 cell monolayers. The metabolic activity of CYP3A4 (midazolam 1′-hydroxylase activity) in the CYP3A4-CPR-HAC/Caco-2 cells was constant from 22 to 35 passages, indicating that HAC vectors conferred sufficient and sustained CYP3A4 activity to CYP3A4-CPR-HAC/Caco-2 cells. The strong relationship between the metabolic extraction ratios (ER) obtained from the CYP3A4-CPR-HAC/Caco-2 cells and calculated intestinal extraction ratios in humans (Eg) from reported intestinal availability (Fg) was found for 17 substrates of CYP3A4 (r² = 0.84). The present study suggests that the CYP3A4-CPR-HAC/Caco-2 cell monolayer can serve as an in vitro tool that facilitates the prediction of intestinal extraction ratio (or availability) in humans.     

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.     

Activation of nicotinamide N-methyltrasferase and increased formation of 1-methylnicotinamide (MNA) in atherosclerosis

Nicotinamide N-methyltrasferase (NMMT) catalyzes the conversion of nicotinamide (NA) to 1-methylnicotinamide (MNA). Recent studies have reported that exogenous MNAexerts anti-thrombotic and anti-inflammatory activity, suggesting that endogenous NMMT-derived MNA may play a biological role in the cardiovascular system.In the present study, we assayed changes in hepatic NNMTactivity and MNAplasma levels along the progression of atherosclerosis in apoE/LDLR^?/? mice, as compared to age-matched wild-type mice. Atherosclerosis progression in apoE/LDLR^?/? mice was quantified in aortic root, while hepatic NNMT activity and MNAplasma concentrations were concomitantly measured in 2-, 3-, 4-, and 6-month-old mice.In apoE/LDLR^?/? mice, atherosclerotic plaques developed in the aortic roots beginning at the age of 3 months and gradually increased in size, macrophage content, and inflammation intensity over time, as detected by Oil-Red O staining, CD68 immunostaining, and in situ zymography (MMP2/MMP9 activity). Hepatic NNMT activity was upregulated approximately two-fold in apoE/LDLR^?/? mice by the age of 2 months, as compared to wild-type mice (1.03 ± 0.14 vs. 0.64 ± 0.23 pmol/min/mg, respectively). MNAplasma concentrations were also elevated approximately two-fold (0.30 ± 0.13 vs. 0.17 ± 0.04 μmol/l, respectively). As atherosclerosis progressed, hepatic NMMTactivity and MNAplasma concentrations increased five-fold in 6-month-old apoE/LDLR^?/? mice at the stage of advanced atherosclerotic plaques (NMMT activity: 2.29 ± 0.34 pmol/min/mg, MNA concentration: 1.083 ± 0.33 μmol/l).In summary, the present study demonstrated that the progression of vascular inflammation and atherosclerosis was associated with the upregulation of hepatic NNMT activity and subsequent increase in endogenous MNAplasma levels. Given the anti-thrombotic and anti-inflammatory properties of exogenous MNA, robust activation of an endogenous NA-MNApathway in atherosclerosis may play an important compensatory role.     

Lipid-soluble green tea extract: Genotoxicity and subchronic toxicity studies

To assess the potential safety of lipid soluble green tea extract, also referred to as lipid soluble tea polyphenols (LSTP), a series of genotoxicity tests were conducted, including an Ames, in vivo mouse micronucleus, and in vivo mouse sperm abnormality test. The toxicity of LSTP was evaluated in 90- and 30-day feeding studies. LSTP did not show mutagenic activity in the Ames test and no genotoxic potential in the in vivo assays at doses up to 10 g/kg body weight (bw). In the 90-day feeding study, LSTP was given in the diet at levels providing 0, 0.125, 0.25, or 0.50 g/kg bw/day. No significant effects were noted on body weight, food consumption, hematology, clinical chemistry, organ weights, and histopathological examination. The no-observed-adverse-effect level (NOAEL) was therefore considered to be 0.50 g/kg bw/day, the highest dose tested. Likewise, dosing of SD rats by gavage for 30 days also showed no adverse effects of growth, hematology, clinical chemistry, organ weights, or histopathology at doses of 0.58, 1.17, and 2.33 g/kg bw/day. The NOAEL in the 30-day study was considered to be the highest dose tested. These data provide evidence to support the safe use of LSTP in food.     

Skin absorption and human exposure estimation of three widely discussed UV filters in sunscreens – In vitro study mimicking real-life consumer habits

Due to health concerns about safety, three UV-filters (Benzophenone-3, BP3, 10%; Ethylhexyl Methoxycinnamate, EHMC, 10%; Butyl Methoxydibenzoylmethane, BMDBM; 5%) were examined in vitro for absorption on full-thickness pig-ear skin, mimicking human in-use conditions. Kinetic profiles confirmed the rapid permeation of BP3; after the first hour of skin (frozen-stored) exposure to 2 mg/cm² (W/O sunscreen; recommended but unrealistic amount), about 0.5% of the applied dose passed into the receptor fluid. The absorption rate of filters was higher from W/O than from O/W emulsions. The fresh/frozen-stored skin permeability coefficient (0.83–0.54) for each UV filter was taken into account. Systemic Exposure Dosage of BP3, EHMC, BMDBM for humans as a consequence of (i) whole-body and (ii) face treatment with 0.5 mg/cm² of W/O sunscreen for 6-h skin exposure followed by washing and subsequent 18-h permeation (a realistic scenario) were estimated to be (i) 4744, 1032 and 1036 μg/kg-bw/day, and (ii) 153, 33 and 34 μg/kg-bw/day, respectively. From Margin of Safety for BP3, EHMC and BMDBM (i) 42, 485 and 192 as well as (ii) 1307; 15,151 and 5882, respectively, only the value of 42 (<100) for BP3 indicated a possible health risk. Escalation of a phobia towards all organic UV filters is undesirable.