Investigating Tm Method Specificity Using Oligonucleotide Sequence Variants.

All starting materials and the active pharmaceutical ingredient (API) of a drug product must be subjected to analytical identity (ID) testing as part of the release prior to their introduction into the pharmaceutical manufacturing process. Generally, it is preferable for Quality Control (QC) laboratories to perform ID tests using a simple and fast to perform, yet highly specific, analytical method. This preference also applies to oligonucleotides, an emerging class of APIs, where a combined ID testing strategy should be applied, including intact mass determination and a sequence-specific method. Within this work, we investigated whether ultravioloet (UV)-spectrometric determination of the melting temperature (Tm) of oligonucleotides is a suitable sequence-specific ID test for these substances in the pharmaceutical routine QC. Therefore, this method was evaluated for its specificity toward deviating oligonucleotide sequences. For this, model oligonucleotide sequences and variants thereof were designed, synthesized, and analyzed, resulting in precise and specific data. Even single base exchanges or single nucleotide deletions and insertions in the sequences led to significant changes in the measured Tm of the corresponding oligonucleotide duplexes. These results indicate a generally high specificity of the method toward subtle changes in oligonucleotide sequences and confirm the applicability of the analytical method as part of the ID testing strategy for oligonucleotides in the pharmaceutical QC environment.

Comparative Ochratoxin Toxicity: A Review of the Available Data.

Ochratoxins are a group of mycotoxins produced by a variety of moulds. Ochratoxin A (OTA), the most prominent member of this toxin family, was first described by van der Merwe et al. in Nature in 1965. Dietary exposure to OTA represents a serious health issue and has been associated with several human and animal diseases including poultry ochratoxicosis, porcine nephropathy, human endemic nephropathies and urinary tract tumours in humans. More than 30 years ago, OTA was shown to be carcinogenic in rodents and since then extensive research has been performed in order to investigate its mode of action, however, this is still under debate. OTA is regarded as the most toxic family member, however, other ochratoxins or their metabolites and, in particular, ochratoxin mixtures or combinations with other mycotoxins may represent serious threats to human and animal health. This review summarises and evaluates current knowledge about the differential and comparative toxicity of the ochratoxin group.

Comparison of two ELISA-based methods for the detection of microcystins in blood serum.

Microcystins (MCs) are cyanobacterial toxins which place the public at risk via exposure to MC contaminated water, food or algal food supplements. Subsequent to the fatal intravenous exposure of dialysis patients in Caruaru, Brazil, several techniques (LC-MS, GC-MS and ELISA) were adapted to detect MCs in human serum. As patients chronically exposed to low concentrations of MCs also present with very low MC serum levels, only LC-MS methodology would appear to allow detection of these MC levels. However, LC-MS detection depends on the availability of respective MC congener standards and the levels of non-covalently bound MC in the sample. In contrast, immunological techniques, e.g. MC-ELISA potentially could detect even covalently bound MC, provided the MC-antibody was raised against an epitope found in nearly all of the MC congeners. As the Adda-side-chain moiety is present in nearly all of the MC congeners known to date, the anti-Adda antibodies, when applied in Adda-ELISAs, could represent a relatively simple and robust technique for the qualitative and quantitative determination of MC in human serum. The aim of the current study was to determine whether commercially available Adda-ELISAs and their respective sample preparation methods would allow MC quantification in human serum. The Adda-ELISA (polyclonal antibody) and the Adda-ELISA (monoclonal antibody) kit for serum (Serum-ELISA) were used for determination of the concentration-dependent recovery of MCs in MC-spiked serum. Human serum samples were spiked with varying concentrations of MCs (MC-LR, -YR, -RR, -LA, -LW, -LF and defined MC mixtures) and extracted using two different methods. MC-spiked bovine serum and standard cell culture medium containing 10% FBS served to investigate potential matrix effects. Inter-laboratory comparison was performed allowing identification of potential sources of error. The results suggest that both ELISAs are suitable tools for the analysis of MCs in human blood serum although both also displayed some weaknesses notably the time needed for sample preparation or the overestimation of some specific MC congener concentrations. Based on the ELISA detection ranges, sample concentration and/or MC spiking may be required for detection of low levels of MCs in human blood.

Pitfalls in microcystin extraction and recovery from human blood serum.

BACKGROUND: Microcystins (MCs) contaminate water bodies due to cyanobacterial blooms all over the world, leading to frequent exposure of humans to MCs through consumption of meat, fish, seafood, blue-green algal products and water, accidental ingestion of contaminated water and scum during recreational activities and inhalation of cyanobacterial aerosols. For monitoring of human exposure, sensitive screening methods are needed. However, during the analytical process of various matrices, such as human serum, some problems appear to regularly occur during sample preparation and storage, leading to MC loss and thus to underestimation of the true MC concentration. The aim of the current study was therefore to assess the pitfalls of the MC-extraction method from human serum with more detail. METHODS: Six MC congeners (MC-LR, -YR, -RR, -LA, -LW, -LF) and defined equimolar MC mixtures thereof were spiked into human serum, and quantified using the commercially available Adda-ELISA subsequent to standard extraction (methanol extraction with subsequent SPE). To detect the potential influence of sample storage and preparation/storage materials, different types of material such as glass, standard polypropylene and surface-treated polypropylene were compared. RESULTS: Loss of MC during preparation and storage is largely dependent on (1) the handling of the stored material, (2) the 'surface' of the storage material and (3) the hydrophobicity of the MCs. CONCLUSIONS: The pitfalls described for MC analysis with the ELISA are primarily associated with sample preparation and clean-up and thus also apply to other analytical techniques for MC detection beyond the ELISA used. It can be concluded that ELISA-based methods are suitable tools for the detection of MCs in human sera and other samples.

Primary porcine proximal tubular cells as an alternative to human primary renal cells in vitro: an initial characterization.

BACKGROUND: A good in vitro model should approximate an in vivo-like behavior as closely as possible in order to reflect most likely the in vivo situation. Regarding renal physiology of different species, humans are more closely related to pigs than to rodents, therefore primary porcine kidney cells (PKC) and their subsequent cell strain could be a valid alternative to primary human cells for renal in vitro toxicology. For this PKC must display inherent characteristics (e.g. structural organization) and functions (e.g. transepithelial transport) as observed under in vivo conditions within the respective part of the kidney. RESULTS: We carried out a comprehensive characterization of PKC and their subsequent cell strain, including morphology and growth as well as transporter expression and functionality. The data presented here demonstrate that PKC express various transporters including pMrp1 (abcc1), pMrp2 (abcc2), pOat1 (slc22a6) and pOat3 (slc22a8), whereas pMdr1 (abcb1) and pOatp1a2 (slco1a2) mRNA could not be detected in either the PKCs or in the porcine cortical tissue. Functionality of the transporters was demonstrated by determining the specific PAH transport kinetics. CONCLUSIONS: On the basis of the presented results it can be concluded that PKC and to some extent their subsequent cell strain represent a valuable model for in vitro toxicology, which might be used as an alternative to human primary cells.

Bioavailability and potential carcinogenicity of polycyclic aromatic hydrocarbons from wood combustion particulate matter in vitro.

Due to increasing energy demand and limited fossil fuels, renewable energy sources have gained in importance. Particulate matter (PM) in general, but also PM from the combustion of wood is known to exert adverse health effects in human. These are often related to specific toxic compounds adsorbed to the PM surface, such as polycyclic aromatic hydrocarbons (PAH), of which some are known human carcinogens. This study focused on the bioavailability of PAHs and on the tumor initiation potential of wood combustion PM, using the PAH CALUX® reporter gene assay and the BALB/c 3T3 cell transformation assay, respectively. For this, both cell assays were exposed to PM and their respective organic extracts from varying degrees of combustion. The PAH CALUX® experiments demonstrated a concentration-response relationship matching the PAHs detected in the samples. Contrary to expectations, PM samples from complete (CC) and incomplete combustion (IC) provided for a stronger and weaker response, respectively, suggesting that PAH were more readily bioavailable in PM from CC. These findings were corroborated via PAH spiking experiments indicating that IC PM contains organic components that strongly adsorb PAH thereby reducing their bioavailability. The results obtained with organic extracts in the cell transformation assay presented the highest potential for carcinogenicity in samples with high PAH contents, albeit PM from CC also demonstrated a carcinogenic potential. In conclusion, the in vitro assays employed emphasize that CC produces PM with low PAH content however with a general higher bioavailability and thus with a nearly similar carcinogenic potential than IC PM.

Investigation of microcystin congener-dependent uptake into primary murine neurons.

BACKGROUND: Contamination of natural waters by toxic cyanobacteria is a growing problem worldwide, resulting in serious water pollution and human health hazards. Microcystins (MCs) represent a group of > 80 cyclic heptapeptides, mediating cytotoxicity via specific protein phosphatase (PP) inhibition at equimolar concentrations (comparable toxicodynamics). Because of the structure and size of MCs, active uptake into cells occurs via organic anion-transporting polypeptides (OATP/Oatp), as confirmed for liver-specific human OATP1B1 and OATP1B3, mouse Oatp1b2 (mOatp1b2), skate Oatp1d1, and the more widely distributed OATP1A2 expressed, for example, at the blood-brain barrier. Tissue-specific and cell-type-specific expression of OATP/Oatp transporters and specific transport of MC congeners (toxicokinetics) therefore appear prerequisite for the reported toxic effects in humans and other species upon MC exposure. Beyond hepatotoxicity induced by the MC-LR congener, the effects of other MC congeners, especially neuronal uptake and toxicity, are unknown. OBJECTIVES: In this study we examined the expression of mOatps and the uptake of congeners MC-LR, MC-LW, and MC-LF in primary murine neurons. METHODS: Intracellular MC accumulation was indicated indirectly via uptake inhibition experiments and directly confirmed by Western blot analysis and a PP inhibition assay. Neuronal mOatp expression was verified at the mRNA and protein level. RESULTS: MCs can cross neuronal cell membranes, with a subsequent decrease of PP activity. Of 15 mOatps, 12 were expressed at the mRNA level, but we found detectable protein levels for only two: mOatp1a5 (Slco1a5) and the known MC-LR transporter mOatp1b2 (Slco1b2). CONCLUSIONS: These data suggest mOatp-mediated uptake of MC congeners into neurons, thus corroborating earlier assumptions of the neurotoxic potential of MCs.

Development and characterization of a monoclonal antibody against Ochratoxin B and its application in ELISA.

A monoclonal antibody specific to ochratoxin B (OTB) was employed for the development of an indirect competitive OTB-ELISA. The optimized OTB-ELISA resulted in a limit of detection (LOD) for OTB of 3 µg/L (8 nM), a limit of quantification (LOQ) of 3.7 µg/L (10 nM), and a 50% inhibitory concentration (IC(50)) of 150 nM. Due to very low cross-reactivity to OTA (2.7%) and structurally related molecules (0%), this OTB-ELISA was found to be suitable to detect OTB with excellent precision in different matrices, i.e., beer, coffee and wine. Therefore, this OTB-ELISA will allow screening of OTB in food and feed products.

Production and characterization of monoclonal antibodies against ochratoxin B.

Monoclonal antibodies against ochratoxin B (OTB) were generated by immunizing Balb/c mice with OTB conjugated to keyhole limpet hemocyanin (KLH) via carbodiimide reactions with CHMC and EDAC. A stable hybridoma cell line 2F1.E10 was produced by fusion of murine splenocytes and myeloma cells. The obtained antibodies were characterized using an indirect competitive ELISA. The detection limit was calculated (27+/-2 nM OTB) and 50% binding inhibition was reached at 500 nM free OTB. A low cross-reactivity to ochratoxin A (OTA) of 3.3% and no cross-reactivities to either coumarin or DL-phenylalanine were observed, suggesting a highly specific OTB antibody. The antibody type was identified as IgG class 1 with the light chain being of the kappa configuration. These antibodies can be used in an indirect competitive ELISA to detect OTB in the nanomolar to micromolar concentration range and may be useful for the analysis of contaminated food items.

Ochratoxin A: comparative pharmacokinetics and toxicological implications (experimental and domestic animals and humans).

The causal factors for the species- and sex-differences associated with ochratoxin-mediated toxicity remain unclear. Variations in kinetic parameters may play a major role in explaining these differences, however, discrepancies and inaccuracies in the toxicokinetics reported in the literature for various species, make comparison and hence the extrapolation to the human situation impossible. The one- and two-compartment open models currently proposed may be insufficient to enable an accurate representation of the actual situation in vivo. It is likely that at least three if not four compartments must be assumed to account for the reported effects. The application of such models to existing raw data would most likely provide for a more accurate base set of toxicokinetic data and contribute to a more accurate human risk assessment. Possible explanations for the reported inconsistencies and their impact on the proposed mechanism(s) of action of OTA and risk assessment are discussed.

Species- and sex-specific variations in binding of ochratoxin A by renal proteins in vitro.

The mycotoxin ochratoxin A (OTA) is a potent renal carcinogen in rodents and induces renal fibrosis in pigs. Furthermore, OTA has been associated with the development of renal tumors and nephropathies in humans. Large species- and sex-differences are observed in sensitivity toward OTA-mediated toxicity and carcinogenicity, yet neither the mechanism(s) resulting in OTA toxicity nor the reasons for the observed species- and sex-specificities are known. This paper investigated variations in OTA handling viz binding to renal proteins which could possibly explain the observed differences in OTA susceptibility in vivo and in vitro. The results obtained via a modification of a standard receptor-binding assay demonstrated the presence of at least one homogeneous binding component in renal cortical homogenates from pig, mouse, rat and humans. This component was shown to bind OTA in a specific and saturable manner. A range of compounds selected for their affinity for steroid receptors and/or for various known organic anion transporters were employed in a competition assay to answer the question whether this homogenous OTA binding component represents a steroid-like receptor component or one of the known organic anion transporters of the kidney. Although many of the compounds were able to compete with OTA for protein-binding, the competition patterns displayed a distinct species specificity and did not correspond to the competition patterns associated with presently known organic anion transporters of the kidney in the mouse, rat or human. The data thus suggests the presence of a new organic anion transporter or more likely, a cytosolic binding component of unknown function with high affinity and capacity for OTA binding in humans, rats, mice and possibly pigs.