Assessing the potential of pharmaceuticals and their transformation products to cause mutagenic effects: Implications for gene expression profiling.

The selection and prioritization of pharmaceuticals and their transformation products for evaluating effects on the environment and human health is a challenging task. One common approach is based on compounds (e.g., mixture composition, concentrations), and another on biology (e.g., relevant endpoint, biological organizational level). Both of these approaches often resemble a Lernaean Hydra-they can create more questions than answers. The present study embraces this complexity, providing an integrated approach toward assessing the potential effects of transformation products of pharmaceuticals by means of mutagenicity, estrogenicity, and differences in the gene expression profiles. Mutagenicity using the tk kinase assay was applied to assess a list of 11 priority pharmaceuticals, namely, atenolol, azithromycin, carbamazepine, diclofenac, ibuprofen, erythromycin, metoprolol, ofloxacin, propranolol, sulfamethoxazole, and trimethoprim. The most mutagenic compounds were found to be ß-blockers. In parallel, the photolabile pharmaceuticals were assessed for their mixture effects on mutagenicity (tk assay), estrogenicity (T47D- KBluc assay), and gene expression (microarrays). Interestingly, the mixtures were mutagenic at the µg/L level, indicating a synergistic effect. None of the photolysed mixtures were statistically significantly estrogenic. Gene expression profiling revealed effects related mainly to certain pathways, those of the p53 gene, mitogen-activated protein kinase, alanine, aspartate, and glutamate metabolism, and translation-related (spliceosome). Fourteen phototransformation products are proposed based on the m/z values found through ultra-performance liquid chromatography-tandem mass spectrometry analysis. The transformation routes of the photolysed mixtures indicate a strong similarity with those obtained for each pharmaceutical separately. This finding reinforces the view that transformation products are to be expected in naturally occurring mixtures. Environ Toxicol Chem 2016;35:2753-2764. © 2016 SETAC.

Arginine residues are important in determining the binding of human monoclonal antiphospholipid antibodies to clinically relevant antigens.

In the antiphospholipid syndrome (APS), antiphospholipid Abs (aPL) bind to anionic phospholipids (PL) and various associated proteins, especially beta(2)-glycoprotein I (beta2GPI) and prothrombin. In the present study, we show that altering specific Arg residues in the H chain of a human pathogenic beta2GPI-dependent aPL, IS4, has major effects on its ability to bind these clinically important Ags. We expressed whole human IgG in vitro by stable transfection of Chinese hamster ovary cells with expression plasmids containing different V(H) and V(L) sequences. V(H) sequences were derived from IS4 by altering the number of Arg residues in CDR3. V(L) sequences were those of IS4, B3 (anti-nucleosome Ab), and UK4 (beta2GPI-independent aPL). Binding of the expressed H/L chain combinations to a range of anionic, neutral, and zwitterionic PL, as well as prothrombin, beta2GPI, dsDNA, and chicken OVA, was determined by ELISA. Of four Arg residues in IS4VH CDR3 substituted to Ser, two at positions 100 and 100g, reduced binding to all Ags, while two at positions 96 and 97 reduced binding to beta2GPI but increased or decreased binding to different PL. Eleven of 14 H/L chain combinations displayed weak binding to OVA with Arg to Ser replacements of all four Arg residues enhancing binding to this Ag. Only one H/L chain combination bound neutral PL and none bound dsDNA; hence, these effects are particularly relevant to Ags important in antiphospholipid syndrome. We hypothesize that these four Arg residues have developed as a result of somatic mutations driven by an Ag containing both PL and beta2GPI.

The critical role of arginine residues in the binding of human monoclonal antibodies to cardiolipin.

Previously we reported that the variable heavy chain region (VH) of a human beta2 glycoprotein I-dependent monoclonal antiphospholipid antibody (IS4) was dominant in conferring the ability to bind cardiolipin (CL). In contrast, the identity of the paired variable light chain region (VL) determined the strength of CL binding. In the present study, we examine the importance of specific arginine residues in IS4VH and paired VL in CL binding. The distribution of arginine residues in complementarity determining regions (CDRs) of VH and VL sequences was altered by site-directed mutagenesis or by CDR exchange. Ten different 2a2 germline gene-derived VL sequences were expressed with IS4VH and the VH of an anti-dsDNA antibody, B3. Six variants of IS4VH, containing different patterns of arginine residues in CDR3, were paired with B3VL and IS4VL. The ability of the 32 expressed heavy chain/light chain combinations to bind CL was determined by ELISA. Of four arginine residues in IS4VH CDR3 substituted to serines, two residues at positions 100 and 100 g had a major influence on the strength of CL binding while the two residues at positions 96 and 97 had no effect. In CDR exchange studies, VL containing B3VL CDR1 were associated with elevated CL binding, which was reduced significantly by substitution of a CDR1 arginine residue at position 27a with serine. In contrast, arginine residues in VL CDR2 or VL CDR3 did not enhance CL binding, and in one case may have contributed to inhibition of this binding. Subsets of arginine residues at specific locations in the CDRs of heavy chains and light chains of pathogenic antiphospholipid antibodies are important in determining their ability to bind CL.