Proposal for a revised Reference Concentration (RfC) for manganese based on recent epidemiological studies

In 1993, based on observations of subclinical neurological effects in workers, the United States Environmental Protection Agency (US EPA) published a Reference Concentration (RfC) of 0.05 μg/m³ for manganese (Mn). The geometric mean exposure concentration, 150 μg/m³ respirable Mn, was considered the lowest observable adverse effect level (LOAEL), and uncertainty factors (UFs) were applied to account for sensitive populations, database limitations, a LOAEL, subchronic exposure, and potential differences in toxicity of different forms of Mn. Based on a review of more recent literature, we propose two alternate Mn RfCs. Of 12 more recent occupational studies of eight cohorts with chronic exposure durations, examining subclinical neurobehavioral effects, predominantly on the motor system, three were considered appropriate for development of an RfC. All three studies yielded no observable adverse effect levels (NOAELs) of approximately 60 μg/m³ respirable Mn. Converting the occupational NOAEL to a human equivalent concentration (HEC) of 21 μg/m³ (for continuous exposure) and applying a UF of 10 to account for intraspecies variability yielded an RfC of 2 μg/m³. We also derived a similar RfC (7 μg/m³) using an Mn benchmark dose (BMD) as the point of departure. Overall confidence in both RfCs is medium.     

Derivation of a chronic reference dose and reference concentration for trimethylbenzenes and C9 aromatic hydrocarbon solvents

Trimethylbenzenes (TMBs) and C9 aromatic hydrocarbon solvents are structurally similar and have similar toxicity. Based on a review of the entire TMB and C9 aromatic hydrocarbon solvents toxicology database, oral and inhalation studies were identified to serve as the basis for a Reference dose (RfD) and Reference concentrations (RfC). The RfD and RfC were derived using standard USEPA methods and assumptions. The RfD was calculated to be 0.4 mg/kg/day using a 90-day oral study that resulted in a NOAEL of 600 mg/kg/day, based on a lack of adverse effects at the highest dose level (reversible effects such as increased serum phosphorus levels and liver and kidney weights), along with a total uncertainty factor of 1000. For the RfC, three studies were considered based on different study designs and toxicological endpoints, including neurotoxicity, systemic toxicity, and potential developmental and reproductive toxicity. For all three studies, as the calculated RfCs were consistent (3–4 mg/m³), the most conservative RfC, 3 mg/m³, was selected. The C9 aromatic hydrocarbon solvents referred to herein are based on chemistries assessed as part of the TSCA Section 4 Test Rule. These solvents contain primarily ethyl toluene and tri-methyl benzene isomers, but the specific compositions can vary based on feedstock and manufacturing process, thus, it is important to consider the composition of any specific solvent to assess similarity to that assessed in the TSCA Section 4 Test Rule program.     

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.     

Manganese exposure through drinking water during pregnancy and size at birth: A prospective cohort study

The essential element manganese (Mn) might be toxic at excess exposure. We assessed the impact of elevated Mn exposure through drinking water during pregnancy on birth size in a population-based cohort(n = 1695) in rural Bangladesh. Concentrations of water Mn (median = 236 μg/L, range = 7.1–6336; n = 1177) and erythrocyte Mn (median = 30 μg/kg, range = 6.3–114; n = 758) were measured using ICP-MS. In regression analyses, newborns of women in the highest tertile of water Mn (median = 1495 μg/L) were 0.49 cm (0.20 SD) shorter (B = −0.42; 95% CI: −0.77, −0.08) than those in the lowest tertile (56 μg/L). The inverse association was significant in girls and also in boys of mothers with lowest hemoglobin values, likely due to higher absorption of Mn. Manganese concentrations in water and erythrocytes did not correlate, and the associations of the latter with birth size were less obvious. This study suggests that consumption of water with highly elevated Mn levels during pregnancy may impair fetal growth.     

Manganese accumulation in membrane fractions of primary astrocytes is associated with decreased γ-aminobutyric acid (GABA) uptake,and is exacerbated by oleic acid and palmitate

Manganese (Mn) exposure interferes with GABA uptake; however, the effects of Mn on GABA transport proteins (GATs) have not been identified. We sought to characterize how Mn impairs GAT function in primary rat astrocytes. Astrocytes exposed to Mn (500 μM) had significantly reduced ³H-GABA uptake despite no change in membrane or cytosolic GAT3 protein levels. Co-treatment with 100 μM oleic or palmitic acids (both known to be elevated in Mn neurotoxicity), exacerbated the Mn-induced decline in ³H-GABA uptake. Mn accumulation in the membrane fraction of astrocytes was enhanced with fatty acid administration, and was negatively correlated with ³H-GABA uptake. Furthermore, control cells exposed to Mn only during the experimental uptake had significantly reduced ³H-GABA uptake, and the addition of GABA (50 μM) blunted cytosolic Mn accumulation. These data indicate that reduced GAT function in astrocytes is influenced by Mn and fatty acids accumulating at or interacting with the plasma membrane.     

Human exposure to acrolein: Time-dependence and individual variation in eye irritation

The aim of the study was to examine the time dependence on sensory irritation detection following exposure to threshold levels of acrolein, in humans. The exposures occurred in an exposure chamber and the subjects were breathing fresh air through a mask that covered the nose and mouth. All participants participated in four exposure conditions, of which three consisted of a mixture of acrolein and heptane and one of only heptane. Exposure to acrolein at a concentration half of the TLV-C lead to sensory irritation. The perceived sensory irritation resulted in both increased detectability and sensory irritation after about 6.8 min of exposure in 58% of the participants. The study confirm the previously suggested LOAEL of about 0.34 mg/m³ for eye irritation due to acrolein exposure. The sensory irritation was still significant 10 min after exposure. These results have implications for risk assessment and limit setting in occupational hygiene.     

Safety evaluation of metal exposure from commonly used moisturizing and skin-lightening creams in Nigeria

The concentrations of ten metals (Cd, Pb, Ni, Cr, Cu, Co, Fe, Mn, Zn and Al) were measured in some commonly used moisturizing and skin-lightening creams in Nigeria with a view to providing information on the risk of exposure to metals from the use of these products. The metal concentrations in these products were measured by atomic absorption spectrometry after acid digestion of the samples. The measured concentrations of metals in the skin moisturizing creams ranged from <0.15 to 6.3 μg/g Cd, <0.02 to 17.5 μg/g Cu, 2.25 to 6.25 μg/g Cr, <0.25 to 124.3 μg/g Al, 0.2 to 7.3 μg/g Pb, <0.03 to 10.7 μg/g Ni, 17.3 to 372.0 μg/g Zn, <0.02 to 1.0 μg/g Co, 17.75 to 28.8 μg/g Mn, <0.1 to 89.8 μg/g Fe while the concentrations of metals in the skin-lightening products ranged from <0.15 to 16.5 μg/g Cd, <0.02 to 10.0 μg/g Cu, 4.25 to 8.0 μg/g Cr, <0.25 to 128.0 μg/g Al, 0.5 to 4.5 μg/g Pb, <0.03 to 1.65 μg/g Ni, 24.7 to 267.5 μg/g Zn, <0.02 to 2.5 μg/g for Co, 19.3 to 31.8 μg/g Mn, 9.5 to 211.63 μg/g Fe. In a significant number (>93%) of the samples investigated the concentrations of Pb, Cd, Ni and Co were below the specified limit, or the maximal limit for impurities in colour additives in cosmetics for external use. However, Cr was found at concentrations above the allergenic limit of 1 μg/g. The results also showed that skin-lightening creams contained higher concentrations of the studied metals than the moisturizing creams, except for Ni, which indicates that persons who uses skin-lightening creams in preference to moisturizing ones, are exposed to higher concentrations of metals.     

Altered sensitivity to nitric oxide donors,induced by intravascular infusion of quantum dots,in murine mesenteric arteries

Quantum dots (QDs) are utilised in imaging diagnostics, tissue engineering and medical therapeutics, however, their influence on vascular function is not ascertained. Here, we examined small mesenteric arterial responses after acute intravascular exposure to QDs. Incubation in mercaptoundecanoic acid (MUA)-coated QDs (at 15 μg/mL) had no influence on endothelial-dependent dilator responses (Acetylcholine; Ach) but led to an attenuated relaxation to the nitric oxide donor, sodium nitroprusside (SNP). Conversely, incubation in POSS-PCU coated QDs (at 15 μg/mL) led to attenuated Ach responses (10^? 11–10^? 3 M; n = 5, P < 0.05), but had no influence on SNP-induced relaxation. At lower concentrations of POSS-PCU coated QDs (5 μg/mL), Ach responses were preserved. We demonstrate that acute exposure to QDs, can attenuate vasodilation but not vasoconstriction, and is dependent on their surface coatings. Our findings have implications in QD use for imaging diagnostics in disease states, where SNP based drugs are used in therapeutic intervention.From the Clinical EditorIn this paper, the influence of quantum dots on vascular function is investigated---an important aspect to consider with the growing utility of quantum dots in imaging diagnostics, tissue engineering and medical therapeutics.     

Zinc oxide nanoparticle disruption of store-operated calcium entry in a muscarinic receptor signaling pathway

The influences of ZnO nanoparticles on cellular responses to activation of muscarinic receptors were studied in Chinese hamster ovary cells expressing the human M3 muscarinic acetylcholine receptor. ZnO particles (20 nm) induced cytotoxicity in a time and concentration-dependent manner: following a 24 h exposure, toxicity was minimal at concentrations below 20 μg/ml but virtually complete at concentrations above 28 μg/ml. ZnO particles did not affect antagonist binding to M3 receptors or allosteric ligand effects, but increased agonist binding affinity while eliminating guanine nucleotide sensitivity. At a noncytotoxic concentration (10 μg/ml), ZnO increased resting [Ca²⁺]_i from 40 to 130 nM without compromising calcium homeostatic mechanisms. ZnO particles had minimal effects on IP3- or thapsigargin-mediated release of intracellular calcium from the endoplasmic reticulum, but strongly inhibited store-operated calcium entry (capacitive calcium entry). The latter effect was seen as (1) a decrease in the plateau phase of the response and (2) a decrease in Ca²⁺ entry upon introduction of calcium to the extracellular medium following thapsigargin-induced depletion of calcium from the endoplasmic reticulum (EC50’s ≈ 2 μg/ml). Thus, ZnO nanoparticles interfere with two specific aspects of the M3 signaling pathway, agonist binding and store-operated calcium entry.     

Derivation of an occupational exposure limit for inorganic borates using a weight of evidence approach

Inorganic borates are encountered in many settings worldwide, spurring international efforts to develop exposure guidance (US EPA, 2004; WHO, 2009; ATSDR, 2010) and occupational exposure limits (OEL) (ACGIH, 2005; MAK, 2011). We derived an updated OEL to reflect new data and current international risk assessment frameworks. We assessed toxicity and epidemiology data on inorganic borates to identify relevant adverse effects. International risk assessment frameworks (IPCS, 2005, 2007) were used to evaluate endpoint candidates: reproductive toxicity, developmental toxicity, and sensory irritation. For each endpoint, a preliminary OEL was derived and adjusted based on consideration of toxicokinetics, toxicodynamics, and other uncertainties. Selection of the endpoint point of departures (PODs) is supported by dose–response modeling. Developmental toxicity was the most sensitive systemic effect. An OEL of 1.6 mg B/m³ was estimated for this effect based on a POD of 63 mg B/m³ with an uncertainty factor (UF) of 40. Sensory irritation was considered to be the most sensitive effect for the portal of entry. An OEL of 1.4 mg B/m³ was estimated for this effect based on the identified POD and an UF of 1. An OEL of 1.4 mg B/m³ as an 8-h time-weighted average (TWA) is recommended.     

Spray freeze drying for dry powder inhalation of nanoparticles

Formulating nanoparticles for delivery to the deep lung is complex and many techniques fail in terms of nanoparticle stability. Spray freeze drying (SFD) is suggested here for the production of inhalable nanocomposite microcarriers (NCM). Different nanostructures were prepared and characterized including polymeric and lipid nanoparticles. Nanoparticle suspensions were co-sprayed with a suitable cryoprotectant into a cooled, stainless steel spray tower, followed by freeze drying to form a dry powder while equivalent compositions were spray dried (SD) as controls. SFD-NCM possess larger specific surface areas (67–77 m²/g) and lower densities (0.02 g/cm³) than their corresponding SD-NCM. With the exception of NCM of lipid based nanocarriers, SFD produced NCM with a mass median aerodynamic diameter (MMAD) of 3.0 ± 0.5 μm and fine particle fraction (FPF ⩽ 5.2 μm) of 45 ± 1.6% with aerodynamic performances similar to SD-NCM. However, SFD was superior to SD in terms of maintaining the particle size of all the investigated polymeric and lipid nanocarriers following reconstitution (S_f/S_i ratio for SFD ≈ 1 versus >1.5 for SD). The SFD into cooled air proved to be an efficient technique to prepare NCM for pulmonary delivery while maintaining the stability of the nanoparticles.     

Antidermatitic Perspective of Hydrocortisone as Chitosan Nanocarriers: An Ex Vivo and In Vivo Assessment Using an NC/Nga Mouse Model

The aim of this study to administer hydrocortisone (HC) percutaneously in the form of polymeric nanoparticles (NPs) to alleviate its transcutaneous absorption, and to derive additional wound-healing benefits of chitosan. HC-loaded NPs had varied particle sizes, zeta potentials, and entrapment efficiencies, when drug-to-polymer mass ratios increased from 1:1 to 1:8. Ex vivo permeation analysis showed that the nanoparticulate formulation of HC significantly reduced corresponding flux [~24 μg/(cm² h)] and permeation coefficient (~4.8 × 10^? 3 cm/h) of HC across the full thickness NC/Nga mouse skin. The nanoparticulate formulation also exhibited a higher epidermal (1610 ± 42 μg/g of skin) and dermal (910 ± 46 μg/g of skin) accumulation of HC than those associated with control groups. An in vivo assessment using an NC/Nga mouse model further revealed that mice treated with the nanoparticulate system efficiently controlled transepidermal water loss [15 ± 2 g/(m² h)], erythema intensity (232 ± 12), dermatitis index (mild), and thickness of skin (456 ± 27 μm). Taken together, histopathological examination predicted that the nanoparticulate system showed a proficient anti-inflammatory and antifibrotic activity against atopic dermatitic (AD) lesions. Our results strongly suggest that HC-loaded NPs have promising potential for topical/transdermal delivery of glucocorticoids in the treatment of AD. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:1063–1075, 2013     

Formulation,optimization and evaluation of transferosomal gel for transdermal insulin delivery

The present study deals with the development of transferosomal gel containing insulin by reverse phase evaporation method for painless insulin delivery for use in the treatment of insulin dependent diabetes mellitus. The effect of independent process variables like ratio of lipids (soya lecithin:cholesterol), ratio of lipids and surfactants, and ratio of surfactants (Tween 80:sodium deoxycholate) on the in vitro permeation flux (μg/cm²/h) of formulated transferosomal gels containing insulin through porcine ear skin was optimized using 2³ factorial design. The optimal permeation flux was achieved as 13.50 ± 0.22 μg/cm²/h with drug entrapment efficiency of 56.55 ± 0.37% and average vesicle diameter range, 625–815 nm. The in vitro insulin permeation through porcine ear skin from these transferosomal gel followed zero-order kinetics (R² = 0.9232–0.9989) over a period of 24 h with case-II transport mechanism. The in vitro skin permeation of insulin from optimized transferosomal gel by iontophoretic influence (with 0.5 mA/cm² current supply) also provided further enhancement of permeation flux to 17.60 ± 0.03 μg/cm²/h. The in vivo study of optimized transferosomal gel in alloxan-induced diabetic rat has demonstrated prolonged hypoglycemic effect in diabetic rats over 24 h after transdermal administration.     

Inhalation exposure or body burden? Better way of estimating risk – An application of PBPK model

We aim to establish a new way for estimating the risk from internal dose or body burden due to exposure of benzene in human subject utilizing physiologically based pharmacokinetic (PBPK) model. We also intend to verify its applicability on human subjects exposed to different levels of benzene. We estimated personal inhalation exposure of benzene for two occupational groups namely petrol pump workers and car drivers with respect to a control group, only environmentally exposed.Benzene in personal air was pre-concentrated on charcoal followed by chemical desorption and analysis by gas chromatography equipped with flame ionization detector (GC-FID). We selected urinary trans,trans-muconic acid (t,t-MA) as biomarker of benzene exposure and measured its concentration using solid phase extraction followed by high performance liquid chromatography (HPLC).Our estimated inhalation exposure of benzene was 137.5, 97.9 and 38.7 μg/m³ for petrol pump workers, car drivers and environmentally exposed control groups respectively which resulted in urinary t,t-MA levels of 145.4 ± 55.3, 112.6 ± 63.5 and 60.0 ± 34.9 μg g⁻¹ of creatinine, for the groups in the same order.We deduced a derivation for estimation of body burden from urinary metabolite concentration using PBPK model. Estimation of the internal dose or body burden of benzene in human subject has been made for the first time by the measurement of t,t-MA as a urinary metabolite using physiologically based pharmacokinetic (PBPK) model as a tool. The weight adjusted total body burden of benzene was estimated to be 17.6, 11.1 and 5.0 μg kg⁻¹ of body weight for petrol pump workers, drivers and the environmentally exposed control group, respectively using this method. We computed the carcinogenic risk using both the estimated internal benzene body burden and external exposure values using conventional method. Our study result shows that internal dose or body burden is not proportional to level of exposure rather have a non-linear relationship. At a higher exposure level such as for occupational exposure of petrol pump workers and drivers, the conventionally estimated risk is higher than risk estimated from internal body burden. Likewise, for environmental exposure the conventional risk estimation predict lower level than estimated in our study. This emphasizes the importance of body burden and to consider it as a key parameter while estimating health risk at varying level of exposure.     

Bilayered Nail Lacquer of Terbinafine Hydrochloride for Treatment of Onychomycosis

The present study aimed to develop bilayered nail lacquer of terbinafine hydrochloride (TH) for treatment of onychomycosis. The composite nail lacquer formed an underlying drug-loaded hydrophilic layer and overlying hydrophobic vinyl layer. The hydrophilic lacquer made of hydroxylpropyl methylcellulose E-15 contained polyethylene glycol 400 (PEG 400) as a drug permeation enhancer. The vinyl lacquer was composed of poly (4-vinyl phenol) as a water- resistant film former. In vitro permeation studies in Franz diffusion cells indicated that the amount of TH permeated across the human cadaver nail in 6 days was 0.32 ± 0.14, 1.12 ± 0.42, and 1.42 ± 0.53 μg/cm² from control (hydrophilic lacquer devoid of PEG 400), monolayer (hydrophilic lacquer alone), and bilayered nail lacquers, respectively. A higher nail drug load was seen in vitro with the bilayered lacquer (0.59 ± 0.13 μg/mg) as compared to monolayer (0.36 ± 0.09 μg/ mg) and control (0.28 ± 0.07 μg/mg) lacquers. The drug loss despite multiple washing was significantly low (p < 0.001) for the bilayered lacquer owing to the protective vinyl coating. Clinical studies demonstrated the efficacy of bilayered lacquer to achieve better drug load in the nail plate (1.27 ± 0.184 μg/mg) compared to monolayer (0.67 ± 0.18 μg/mg) and control (0.21 ± 0.04 μg/mg) lacquers.     

Nitric oxide mediated effects on reproductive toxicity caused by carbon disulfide in male rats

This study investigated nitric oxide (NO) mediation of carbon disulfide (CS₂) toxicity that compromised male rat spermatogenesis and endocrine function. Rats were exposed to multiple levels of CS₂ concentration (0, 50, 250, 1250 mg/m³). A 1250 mg/m³ CS₂ + sodium nitroprusside (SNP) group and a 1250 mg/m³ CS₂ + NG-monomethyl-l-arginine (l-NMMA) group were established to explore the role of NO in mediating CS₂ toxicity. NO concentrations, NO synthase (NOS) activity, and sex hormone levels were measured, and sperm characteristics were observed and analyzed. Our data show that CS₂ exposure decreased: NOS activity; tissue NO concentrations; serum levels of gonadotropin-releasing hormones, luteinizing hormones, and testosterone; and sperm count and activity. In contrast, increased serum follicle-stimulating hormone concentrations and teratospermia were observed with CS₂ exposure. SNP reduced some of the toxic effects of CS₂, while l-NMMA treatment showed no effect. The results suggests that NO mediates compromised reproductive system function caused by CS₂ exposure.     

Poorly soluble particulates: searching for a unifying denominator of nanoparticles and fine particles for DNEL estimation

Under the new European chemicals regulation, REACH (Registration, Evaluation, Authorization and Restriction of Chemicals) a Derived No-Effect Level (DNEL), i.e., the level of exposure above which humans should not be exposed, is defined. The focus of this paper is to develop a weight-of-evidence-based DNEL-approach for inhaled poorly soluble particles. Despite the common mode of action of inhaled insoluble, spherical particulate matter (PM), a unifying, most appropriate metric conferring pulmonary biopersistence and toxicity has yet not been demonstrated. Nonetheless, there is compelling evidence from repeated rat inhalation exposure studies suggesting that the particle displacement volume is the most prominent unifying denominator linking the pulmonary retained dose with toxicity. Procedures were developed to analyze and model the pulmonary toxicokinetics from short-term to long-term exposure. Six different types of poorly soluble nano- to submicron PMs were compared: ultrafine and pigmentary TiO₂, synthetic iron oxide (Fe₃O₄, magnetite), two aluminum oxyhydroxides (AlOOH, Boehmite) with primary isometric particles approximately of either 10 or 40 nm, and MWCNT. The specific agglomerate densities of these materials ranged from 0.1 g/cm³ (MWCNT) to 5 g/cm³ (Fe₃O₄). Along with all PM, due to their long retention half-times and associated biopersistence in the lung, even short-term inhalation studies may require postexposure periods of at least 3 months to reveal PM-specific dispositional and toxicological characteristics. This analysis provides strong evidence that pulmonary toxicity (sustained inflammation) is dependent on the volume-based cumulative lung exposure dose. Lung toxicity, evidenced by PMN in BAL occurred at lung doses exceeding 10-times the overload threshold. Furthermore, the conclusion is supported that repeated inhalation studies on rats should utilize an experimental window of cumulative volume loads of respirable PM in the range of 1 μl/lung (no-adverse-effect range); however, not exceeding ≈10 μl/lung that would lead to retention half-times increasing 1 year. This can be targeted best by computational toxicology, i.e., the modeling of particle deposition and lung retention biokinetics during the exposure and recovery periods. Inhalation studies exceeding that threshold volume may lead to meaningless findings difficult to extrapolate to any real-life scenario. In summary, this analysis supports a volume-based generic mass concentration of 0.5 μl PM_respirable/m³ × agglomerate density, independent on nano- or submicron-sized properties, as a generic no-adverse effect level in both rats and humans.     

Life-cycle exposure to BDE-47 results in thyroid endocrine disruption to adults and offsprings of zebrafish (Danio rerio)

2,2,4′,4′-Tetrabromodi-phenyl ether (BDE-47) is predominantly concentrated in humans and wildlife and disturbs thyroid hormone homeostasis. The purpose of this study was to characterize the thyroid endocrine disruption induced by life-cycle exposure to BDE-47 in adults and offspring of zebrafish (Danio rerio). We exposed zebrafish embryos at the blastula stage to different concentrations of BDE-47 (1, 5, and 10 μg/L). Exposure duration was 180 days until fish reached adulthood. In F0 larvae, exposure decreased survival and increased malformations at 4 dpf. Thyroid hormone concentrations did not differ significantly between the F0 larvae and controls. All exposures significantly up-regulated expression of tshß, pa8, ugt1 and tg and down-regulated ttr. Significant up-regulation of dio2 and crh was observed in the 10 μg/L BDE-47 group. There was no significant difference in the growth and somatic index between F0 adults and controls. BDE-47 (10 μg/L) significantly decreased whole-body content of thyroxine (T4) but significantly increased triiodothyronine (T3) in both sexes. All exposures up-regulated expression of crh, tshß, pa8, ugt1 and tg and down-regulated ttr. Exposure to 10 μg/L BDE-47 significantly up-regulated dio2 and ugt1 in both sexes. BDE-47 exposure (5 and 10 μg/L) significantly increased the activity of pethoxy-resorufin-O-deethylase and UDP-glucuronosyl transferase. BDE-47 (10 μg/L) significantly increased activity of ethoxy- and methoxy-resorufin-O-deethylase. In F1 offspring without continued BDE-47 (10 μg/L) treatment, T4 significantly decreased and T3 increased. T4 was further decreased and T3 was further increased with continued BDE-47 treatment. Continued BDE-47 exposure decreased hatching and increased malformation compared with those without BDE-47 exposure. Expression of crh, tshß, dio2, pa8, ugt1 and tg was significantly up-regulated without BDE-47 exposure and with continued exposure. With continued BDE-47 exposure, dio1 was significantly up-regulated and ttr was significantly down-regulated. All the genes showed clear differences between continued exposure to 10 μg/L BDE-47 and without BDE-47 exposure. These results suggest that parental exposure to BDE-47 results in thyroid endocrine disruption in adults and offspring.     

Microneedle-assisted delivery of verapamil hydrochloride and amlodipine besylate

The aim of this project was to study the effect of stainless steel solid microneedles and microneedle rollers on percutaneous penetration of verapamil hydrochloride and amlodipine besylate.Verapamil, 2-(3,4-dimethooxyphenyl)-5-[2-(3,4 dimethoxyphenyl)ethyl-methyl-amino]-2-propan-2-yl-pentanenitrile is a calcium channel blocker agent that regulates high blood pressure by decreasing myocardial contractilty, heart rate and impulse conduction. Amlodipine, (R, S)-2-[(2-aminoethoxy) methyl]-4-(2-chlorophenyl)-3-ethoxycarbonyl-5-methoxycarbonyl-6-methyl-1, 4-dihydropyridine, is a calcium channel blocker that is used for the management of hypertension and ischemic heart disease. Passive penetration of verapamil and amlodipine across the skin is low. In vitro studies were performed with microneedle-treated porcine ear skin using vertical static Franz diffusion cells (PermeGear, Hellertown, PA, USA). The receiver chamber contained 5 ml of PBS (pH7.4) and was constantly maintained at 37 °C temperature with a water circulation jacket. The diffusion area of the skin was 1.77 cm². The donor compartment was loaded with 1 ml of the solution containing 2.5 mg/ml of amlodipine besylate. The donor chamber was covered with parafilm to avoid evaporation. Passive diffusion across untreated porcine skin served as control. Aliquots were taken every 2 h for 12 h and analyzed by liquid chromatography–mass spectrometry. Transcutaneous flux of verapamil increased significantly from 8.75 μg/cm²/h to 49.96 μg/cm²/h across microneedle-roller treated porcine skin. Percutaneous flux of amlodipine besylate following the use of stainless steel microneedles was 22.39 μg/cm²/h. Passive flux for the drug was 1.57 μg/cm²/h. This enhancement of amlodipine flux was statistically significant. Transdermal flux of amlodipine with microneedle roller was 1.05 μg/cm²/h in comparison with passive diffusion flux of 0.19 μg/cm²/h. The difference in flux values was also statistically significant. Stainless steel solid microneedles and microneedle rollers increased percutaneous penetration of verapamil hydrochloride and amlodipine besylate. It may be feasible to develop transdermal microneedle patches for these drugs.     

In vivo and in vitro evaluation of the estrogenic properties of the 17β-(butylamino)-1,3,5(10)-estratrien-3-ol (buame) related to 17β-estradiol

BackgroundBuame [17β-(butylamino)-1,3,5(10)-estratrien-3-ol] possesses anticoagulant and antiplatelet activities that are potentially antithrombotic. Since its estrogenicity is unknown, it was evaluated by established methods.MethodsBuame (10, 100, 500, and 1,000 μg/kg), 17β-estradiol (E₂) (100 μg/kg), or propylene glycol (10 ml/kg) were subcutaneously (sc) administered for three days to immature Wistar female rats (21 days old). The relative uterotrophic effect to E₂ was 78 (E₂ = 100) with a relative uterotrophic potency of 1.48 (E₂ = 100). Adult ovariectomized Wistar rats received an sc injection at 8:00 h (reversed cycle) of: 7.5 μg of E₂ (≈ 30 μg/kg), buame (≈ 750, 1,500, 3,000 μg/kg), or corn oil (≈ 1.2 ml/kg). After 24 h, progesterone (4–5 mg/kg) was administered. Sexual receptivity was assessed 5 to 7 h later, and the lordosis quotient (LQ; number lordosis/number mounts × 100) was evaluated.ResultsBuame induced lordosis (LQmax 85 ± 9; ED50 952 ± 19 μg/kg) and E₂ LQmax 56 ± 8; ED50 10 ± 2 μg/kg; the relative LQpotency was 0.51 (E₂ = 100). Buame competed with [³H]E₂ for the estrogen receptor (Buame RBA = 0.15 and Ki = 5.9 × 10^?7 M; E₂ RBA = 100; Ki = 6.6 × 10^?9 M). Buame increased MCF-7 cells proliferation, from 10^?11 to 10^?9 M, its proliferative effect was 1.73–1.79 (E₂ = 3.0–3.9); its relative proliferative effect to E₂ was 33–40% (E₂ = 100%) and relative potency 10.4–10.7 (E₂ = 100). Tamoxifen and fulvestrant (ICI 182,780) inhibited buame's proliferation indicating mediation through estrogen receptors in this response.ConclusionBuame is therefore an estrogen partial agonist with a weak estrogenic activity.