Pilot study of jadomycin B pharmacokinetics and anti-tumoral effects in zebrafish larvae and mouse breast cancer xenograft models.

Despite numerous therapeutic options, multidrug resistance (MDR) remains an obstacle to successful breast cancer therapy. Jadomycin B, a natural product derived from Streptomyces venezuelae ISP5230, maintains cytotoxicity in MDR human breast cancer cells. Our objectives were to evaluate the pharmacokinetics, toxicity, anti-tumoral, and anti-metastatic effects of jadomycin B in zebrafish larvae and mice. In a zebrafish larval xenograft model, jadomycin B significantly reduced the proliferation of human MDA-MB-231 cells at or below its maximum tolerated dose (40 µm). In female Balb/C mice, a single intraperitoneal dose (6 mg/kg) was rapidly absorbed with a maximum serum concentration of 3.4 ± 0.27 µm. Jadomycin B concentrations declined biphasically with an elimination half-life of 1.7 ± 0.058 h. In the 4T1 mouse mammary carcinoma model, jadomycin B (12 mg/kg every 12 h from day 6 to 15 after tumor cell injection) decreased primary tumor volume compared to vehicle control. Jadomycin B-treated mice did not exhibit weight loss, nor significant increases in biomarkers of impaired hepatic (alanine aminotransferase) and renal (creatinine) function. In conclusion, jadomycin B demonstrated a good safety profile and provided partial anti-tumoral effects, warranting further dose-escalation safety and efficacy studies in MDR breast cancer models.

New insights in copper handling strategies in the green alga Chlamydomonas reinhardtii under low-iron condition.

Copper (Cu) is a redox-active transition element critical to various metabolic processes. These functions are accomplished in tandem with Cu-binding ligands, mainly proteins. The main goal of this work was to understand the mechanisms that govern the intracellular fate of Cu in the freshwater green alga, Chlamydomonas reinhardtii, and more specifically to understand the mechanisms underlying Cu detoxification by algal cells in low-Fe conditions. We show that Cu accumulation was up to 51-fold greater for algae exposed to Cu in low-Fe medium as compared to the replete-Fe growth medium. Using the stable isotope 65Cu as a tracer, we studied the subcellular distribution of Cu within the various cell compartments of C. reinhardtii. These data were coupled with metallomic and proteomic approaches to identify potential Cu-binding ligands in the heat-stable proteins and peptides fraction of the cytosol. Cu was mostly found in the organelles (78%), and in the heat-stable proteins and peptides (21%) fractions. The organelle fraction appeared to also be the main target compartment of Cu accumulation in Fe-depleted cells. As Fe levels in the medium were shown to influence Cu homeostasis, we found that C. reinhardtii can cope with this additional stress by utilizing different Cu-binding ligands. Indeed, in addition to expected Cu-binding ligands such as glutathione and phytochelatins, 25 proteins were detected that may also play a role in the Cu-detoxification processes in C. reinhardtii. Our results shed new light on the coping mechanisms of C. reinhardtii when exposed to environmental conditions that induce high rates of Cu accumulation.

Comprehensive In Vitro Metabolism Study of Bisphenol A Using Liquid Chromatography-High Resolution Tandem Mass Spectrometry.

Bisphenol A (BPA) metabolism has been investigated using several in vitro models, including human and rat liver microsomes and subcellular (S9) fractions, as well as human-recombinant cytochrome P450 3A4 (CYP3A4) expressed in Supersomes, for a comprehensive look at all possible metabolic pathways. By an untargeted approach using liquid chromatography coupled to a high-resolution quadrupole-time-of-flight mass spectrometer, we were able to detect a large number of known Phase I and Phase II metabolites of BPA, as well as several previously uncharacterized ones. A detailed fragmentation study of BPA and its detected metabolites was crucial to confirm structures. Isotope-labeled BPA analogs were highly useful for the structural elucidation of many metabolites. These results contribute to a better understanding of BPA metabolism, including pathways that may introduce additional toxicity, as well as help with the assessment of BPA exposure in different biological matrices.

In vitro metabolism of sunscreen compounds by liquid chromatography/high-resolution tandem mass spectrometry.

RATIONALE: Exposure to UV light can induce adverse effects on human health, such as photo-aging, immunosuppression, and cancer. Sunscreens are used to prevent the absorption of UV rays, but certain UV-filtering compounds have been shown to disrupt endocrine systems or act as carcinogens. To assess the effects of the exposure to such compounds, it is important to study the pathways by which they are biotransformed in the body. METHODS: Liquid chromatography coupled to high-resolution tandem mass spectrometry (LC/HRMS/MS) was employed to evaluate the oxidative metabolism and, specifically, the formation of reactive metabolites of six active ingredients commonly used in sunscreen formulations: oxybenzone, avobenzone, homosalate, octisalate, octocrylene, and octinoxate. In vitro incubations were performed with human and rat liver microsomes in the presence of ß-nicotinamide adenine dinucleotide phosphate and glutathione. An LC/HRMS/MS method was developed to identify metabolites employing a biphenyl reversed-phase column for separating parent molecules, metabolites, and glutathione (GSH) adducts. RESULTS: Each tested compound resulted in the formation of several metabolites, including at least one GSH adduct. Compounds containing ester groups were hydrolyzed, and some metabolites of the free acid forms were also detected. High-resolution MS/MS data was crucial for the structural elucidation of metabolites and GSH adducts. Fragmentation pathways were proposed for all parent compounds, as well as each described metabolite and adduct. CONCLUSIONS: The results of this study will help better understand the metabolism and detoxification pathways of these xenobiotics.

Effects of oxidative post-translational modifications on structural stability and self-assembly of λ6 immunoglobulin light chain.

Light chain amyloidosis (AL) originates from the deposition of immunoglobulin light chains (LCs) as amyloid fibrils in the extracellular space of vital organs. Although non-enzymatic post-translational modifications (PTMs) have been shown to contribute to protein misfolding diseases, little is known about their contributions to LC amyloidogenicity. In this study, we investigated the effects of three oxidative PTMs, carbonylation by hydroxynonenal (HNE), oxidation and nitration, on the structure, thermodynamic stability and self-assembly propensity of a LC variable domain from the λ6 germline, Wil. We initially identified the specific residues that are susceptible to oxidative chemical modifications. HNE-conjugation at specific His residues and nitration of Tyr side chains modulated the conformational conversion driving Wil self-assembly and fibrillar aggregates formation. This study reinforces the notion that not only the thermodynamic stability, but also the chemical and structural properties, should be considered when evaluating the amyloidogenic potential of a LC.

A comparison of MS/MS-based, stable-isotope-labeled, quantitation performance on ESI-quadrupole TOF and MALDI-TOF/TOF mass spectrometers.

The peptide-based quantitation accuracy and precision of LC-ESI (QSTAR Elite) and LC-MALDI (4800 MALDI TOF/TOF) were compared by analyzing identical Escherichia coli tryptic digests containing iTRAQ-labeled peptides of defined abundances (1:1, 2.5:1, 5:1, and 10:1). Only 51.4% of QSTAR spectra were used for quantitation by ProteinPilot Software versus 66.7% of LC-MALDI spectra. The average protein sequence coverages for LC-ESI and LC-MALDI were 24.0 and 18.2% (14.9 and 8.4 peptides per protein), respectively. The iTRAQ-based expression ratios determined by ProteinPilot from the 57 467 ESI-MS/MS and 26 085 MALDI-MS/MS spectra were analyzed for measurement accuracy and reproducibility. When the relative abundances of peptides within a sample were increased from 1:1 to 10:1, the mean ratios calculated on both instruments differed by only 0.7-6.7% between platforms. In the 10:1 experiment, up to 64.7% of iTRAQ ratios from LC-ESI MS/MS spectra failed S/N thresholds and were excluded from quantitation, while only 0.1% of the equivalent LC-MALDI iTRAQ ratios were rejected. Re-analysis of an archived LC-MALDI sample set stored for 5 months generated 3715 MS/MS spectra for quantitation, compared with 3845 acquired originally, and the average ratios differed by only 3.1%. Overall, MS/MS-based peptide quantitation performance of offline LC-MALDI was comparable with on-line LC-ESI, which required threefold less time. However, offline LC-MALDI allows the re-analysis of archived HPLC-separated samples.