In silico prediction of dermal absorption of pesticides - an evaluation of selected models against results from in vitro testing.

Current guidance for the estimation of dermal absorption (DA) of pesticides recommends the use of default values, read-across of information between formulations and in vitro testing. While QSARs exist to estimate percutaneous absorption, their use is currently not encouraged. Therefore, the potential of publicly available models for DA estimation was investigated based on data from 564 human in vitro DA experiments on pesticides. The classic Potts Guy model, the correction of Cleek Bunge for highly lipophilic chemicals, the mechanistic model of Mitragotri, and the COSMOS model were used to estimate the permeability coefficient kp. Different approaches were explored to calculate the percentage of external dose absorbed. IH SkinPerm was examined as stand-alone model. The models generally failed to accurately predict experimental values. For 30-40% of the predictions, there was overestimation by one order of magnitude. Three models underpredicted >10% of the cases, the remaining models <5%. DA of hydrophilic substances was typically underpredicted. Overprediction was more prominent for solid preparations and suspensions. The molecular weight, irritation potential and skin thickness did not correlate with the models' predictivity. Of the models investigated, IH SkinPerm performed best with 38% of the predictions within one order of magnitude and 2% underpredicted cases.

Combination effects of azole fungicides in male rats in a broad dose range.

Two 28-day feeding studies were performed in male rats to investigate combination effects of azole fungicides in a broad dose range. Following separate administration of cyproconazole, epoxiconazole, prochloraz, propiconazole, and tebuconazole at five dose levels, the first three compounds were selected to be administered in two different mixtures at three dose levels including very low doses. Here we present the data obtained by clinical observations, pathology, histopathology, clinical chemistry and haematology. The liver was the common main target organ of all compounds and their mixtures. In addition, epoxiconazole exhibited an effect on the adrenals. Furthermore, food consumption and efficiency and body weight (gain) were affected. Adverse effects of the combinations were observed at dose levels at which the individual substances caused similar effects. No evidence of adverse effects was found at dose levels below the previously established NOAELs. Our findings indicate that the concept of dose additivity appears sufficiently protective for risk assessment of the fungicides examined. Besides toxicological testing, tissue residues of the azole compounds in liver, testis and kidney were determined revealing remarkable differences following administration of the single substances and of the mixtures.

Hepatotoxic effects of (tri)azole fungicides in a broad dose range.

The toxicological relevance of effects observed at molecular stage, which occur at dose levels well below classical no-observed adverse effect levels is currently subject to controversial scientific debate. While the importance of molecular effects for the identification of a mode of action or an adverse outcome pathway is undisputed, their impact for other regulatory purposes remains uncertain. Here, we report the results of a 28-day rat-feeding study including three widely used hepatotoxic (tri)azole fungicides (cyproconazole, epoxiconazole and prochloraz) administered individually at five dose levels, ranging from slightly above the reference values to a clear toxic effect dose. Parameters analysed included pathology, histopathology, clinical chemistry and particularly effects on the molecular level. Since azole fungicides are considered to cause liver toxicity by a mechanism involving the constitutive androstane receptor (CAR), a known CAR activator (phenobarbital, PB) was administered to investigate potential similarities between triazoles and PB-mediated liver toxicity by pathway-focused gene expression analysis. Our results show an increase in liver weights and additionally histopathological changes (hepatocellular hypertrophy) for all substances at the top dose levels. The effects on liver weight were most pronounced for cyproconazole by which also the animals receiving the next lower dose were affected. In addition, vacuolisation of hepatocytes was observed at the top dose level. No such findings were obtained with any substance at lower doses to which consumers and operators might be exposed to. In contrast, the expression of sensitive marker genes (like some cytochrome-P-450 isoforms) was significantly affected also at the lower dose levels. While some of these changes, like the induction of genes related to fatty acid and phospholipid metabolism (e.g. Fasn, Fat/Cd36, Ppargc1a) or xenobiotic metabolism (Cyp1a1, Cyp2b1, Cyp3a2), could be associated with high dose effects like hepatocellular vacuolisation or hypertrophy, a histopathological correlate was lacking for others.

Subcellular localization and distribution of the reduced folate carrier in normal rat tissues.

The reduced folate carrier (Rfc1; Slc19a1) mediated transport of reduced folates and antifolate drugs such as methotrexate (MTX) play an essential role in physiological folate homeostasis and MTX cancer chemotherapy. As no systematic reports are as yet available correlating Rfc1 gene expression and protein levels in all tissues crucial for folate and antifolate uptake, storage or elimination, we investigated gene and protein expression of rat Rfc1 (rRfc1) in selected tissues. This included the generation of a specific anti-rRfc1 antibody. Rabbits were immunised with isolated rRfc1 peptides producing specific anti-rRfc1 antiserum targeted to the intracellular C-terminus of the carrier. Using RT-PCR analysis, high rRfc1 transcript levels were detected in colon, kidney, brain, thymus, and spleen. Moderate rRfc1 gene expression was observed in small intestine, liver, bone marrow, lung, and testes whereas transcript levels were negligible in heart, skeletal muscle or leukocytes. Immunohistochemical analyses revealed strong carrier expression in the apical membrane of tunica mucosa epithelial cells of small intestine and colon, in the brush-border membrane of choroid plexus epithelial cells or in endothelial cells of small vessels in brain and heart. Additionally, high rRfc1 protein levels were localized in the basolateral membrane of renal tubular epithelial cells, in the plasma membrane of periportal hepatocytes, and sertoli cells of the testes. Taken together, our results demonstrated that rRfc1 is expressed almost ubiquitously but to very different levels. The predominant tissue distribution supports the essential role of Rfc1 in physiological folate homeostasis. Moreover, our results may contribute to understand antifolate pharmacokinetics and selected organ toxicity associated with MTX chemotherapy.

Stabilisation by freeze-drying of cationically modified silica nanoparticles for gene delivery.

Core shell silica particles with a hydrodynamic diameter of 28nm, an IEP of 7.1 and a zeta potential of +35mV at pH 4.0 were synthesised. The role of freeze-drying for the conservation of zwitterionic nanoparticles and the usefulness of different lyoprotective agents (LPA) for the minimisation of particle aggregation were studied. The activity of the nanoparticles was measured as DNA-binding capacity and transfection efficiency in Cos-1 cells before and after lyophilisation. It was found that massive aggregation occurred in the absence of LPA. Of the various LPAs screened in the present investigations, trehalose and glycerol were found to be well suited for conservation of cationically modified silica nanoparticles with simultaneous preservation of their DNA-binding and transfection activity in Cos-1 cells.

Lectin-mediated drug delivery: discrimination between cytoadhesion and cytoinvasion and evidence for lysosomal accumulation of wheat germ agglutinin in the Caco-2 model.

Lectin-mediated drug delivery may become a promising strategy to improve the efficacy of poorly permeable drugs by utilising active high-capacity transport pathways of epithelial tissues. This requires the elucidation of the basic mechanisms of lectin uptake prior to their practical use. We studied the interaction between the dietary lectin wheat germ agglutinin (WGA) and Caco-2 cells (single cells and monolayers) by a newly established assay design that is able to discriminate between cellular binding and uptake as well as by confocal microscopy: (i) All binding sites available for WGA at the cell membrane were occupied within 10 min of incubation. (ii) Cytoadhesion was followed by immediate uptake. After 20 min, 60% (single cells) or 30% (monolayers) of the membrane bound lectin were internalised. However, regardless of cell arrangement, 80% of the surface bound lectin was taken up into the cells during the course of the experiment. (iii) About 50% of the internalised lectin accumulated within the lysosomes after 1 h. This was confirmed by assays in the presence of monensin, an inhibitor of endosomal acidification, and by colocalisation with lysosomal cathepsin followed by semiquantitative image analysis. Further analysis by immunocytochemistry suggested that the trans-Golgi complex and the caveoli were not involved. Due to cytoadhesion, cytoinvasion and partial lysosomal accumulation, WGA-mediated drug delivery may provide for improved intracellular availability of conjugated drugs or colloidal carrier systems.

Cationic solid-lipid nanoparticles can efficiently bind and transfect plasmid DNA.

The suitability of cationically modified solid-lipid nanoparticles (SLN) as a novel transfection agent was investigated. SLN were produced by hot homogenisation using either Compritol ATO 888 or paraffin as matrix lipid, a mixture of Tween 80 and Span 85 as tenside and either EQ1 (N,N-di-(beta-steaorylethyl)-N,N-dimethylammonium chloride) or cetylpyridinium chloride as charge carrier. The resulting particles were approximately 100 nm in size and showed zeta potentials around +40 mV at pH 7.4. DNA binding was tested by agarose gel electrophoresis. The resulting SLN-DNA complexes were further characterised by AFM and zeta potential measurements. Only the SLN batch SII-13, composed of 4% Compritol, 4% Tween/Span and 1% EQ1, was able to form stable complexes with DNA. Typical complexes were 300 to 800 nm in size. Cytotoxicity and transfection efficiency was tested in vitro on Cos-1 cells. Cationic SLN produced by modification with EQ1 were well tolerated, with LD50 values >3 mg/ml in the LDH release assay and >0.6 mg/ml in the WST-1 assay. Further, SLN-DNA complexes containing between 10 and 200 weight equivalents of SII-13 (matrix lipid) efficiently transfected the galactosidase expression plasmid pCMVbeta in the absence and presence of the endosomolytic agent chloroquine.

A nonviral DNA delivery system based on surface modified silica-nanoparticles can efficiently transfect cells in vitro.

Diverse polycationic polymers have been used as nonviral transfection agents. Here we report the ability of colloidal silica particles with covalently attached cationic surface modifications to transfect plasmid DNA in vitro and make an attempt to describe the structure of the resulting transfection complexes. In analogy to the terms lipoplex and polyplex, we propose to describe the nanoparticle-DNA complexes by the term "nanoplex". Three batches, Si10E, Si100E, and Si26H, sized between 10 and 100 nm and with zeta potentials ranging from +7 to +31 mV at pH 7.4 were evaluated. The galactosidase expression plasmid DNA pCMVbeta was immobilized on the particle surface and efficiently transfected Cos-1 cells. The transfection activity was accompanied by very low cytotoxicity, with LD(50) values in the milligrams per milliliter range. The most active batch, Si26H, was produced by modification of commercially available silica particles with N-(6-aminohexyl)-3-aminopropyltrimethoxysilane, yielding spherical nanoparticles with a mean diameter of 26 nm and a zeta potential of +31 mV at pH 7.4. Complexes of Si26H and pCMVbeta plasmid DNA formed at w/w ratios of 10 were most effective in promoting transfection of Cos-1 cells in the absence of serum. At this ratio, >90% of the DNA was associated with the particles, yielding nanoplexes with a net negative surface charge. When the transfection medium was supplemented with 10% serum, maximum gene expression was observed at a w/w ratio of 30, at which the resulting particle-DNA complexes possessed a positive surface charge. Transfection was strongly increased in the presence of 100 microM chloroquine in the incubation medium and reached approximately 30% of the efficiency of a 60 kDa polyethylenimine. In contrast to polyethylenimine, no toxicity was observed at the concentrations required. Atomic force microscopy of Si26H-DNA complexes revealed a spaghetti-meatball-like structure. The surface of complexes prepared at a w/w ratio of 30 was dominated by particles half-spheres. Complex sizes correlated well with those determined previously by dynamic light scattering.

Silica nanoparticles modified with aminosilanes as carriers for plasmid DNA.

We synthesised silica nanoparticles (SiNP) with covalently linked cationic surface modifications and demonstrated their ability to electrostatically bind, condense and protect plasmid DNA. These particles might be utilised as DNA carriers for gene delivery. All nanoparticles were sized between 10 and 100 nm and displayed surface charge potentials from +7 to +31 mV at pH 7.4. They were produced by modification of commercially available (IPAST) or in-house synthesised silica particles with either N-(2-aminoethyl)-3-aminopropyltrimethoxysilane or N-(6-aminohexyl)-3-aminopropyltrimethoxysilane. All particles formed complexes with pCMVbeta plasmid DNA as evidenced by ratio dependent retardation of DNA in the agarose gel and co-sedimentation of soluble DNA with nanoparticles. High salt and alkaline pH did inhibit complex formation. Absorption onto the particles also decreased the hydrodynamic dimensions of plasmid DNA as shown by photon correlation spectroscopy. Complexes formed in water at a w/w ratio of Si26H:DNA (pCMVbeta) of 300 were smallest with a mean hydrodynamic diameter of 83 nm. For effective condensation a w/w ratio of Si26H:DNA of 30 was sufficient. Further, the absorbed DNA was protected from enzymatic degradation by DNase I.