Rebecca L. Cordell

Curcumin-induced mitotic arrest is characterized by spindle abnormalities, defects in chromosomal congression and DNA damage

The chemopreventive agent curcumin has anti-proliferative effects in many tumour types, but characterization of cell cycle arrest, particularly with physiologically relevant concentrations, is still incomplete. Following oral ingestion, the highest concentrations of curcumin are achievable in the gut. Although it has been established that curcumin induces arrest at the G2/M stage of the cell cycle in colorectal cancer lines, it is not clear whether arrest occurs at the G2/M transition or in mitosis. To elucidate the precise stage of arrest, we performed a direct comparison of the levels of curcumin-induced G2/M boundary and mitotic arrest in eight colorectal cancer lines (Caco-2, DLD-1, HCA-7, HCT116p53+/+, HCT116p53–/–, HCT116p21–/–, HT-29 and SW480). Flow cytometry confirmed that these lines underwent G2/M arrest following treatment for 12h with clinically relevant concentrations of curcumin (5–10 μM). In all eight lines, the majority of this arrest occurred at the G2/M transition, with a proportion of cells arresting in mitosis. Examination of the mitotic index using fluorescence microscopy showed that the HCT116 and Caco-2 lines exhibited the highest levels of curcumin-induced mitotic arrest. Image analysis revealed impaired mitotic progression in all lines, exemplified by mitotic spindle abnormalities and defects in chromosomal congression. Pre-treatment with inhibitors of the DNA damage signalling pathway abrogated curcumin-induced mitotic arrest, but had little effect at the G2/M boundary. Moreover, pH2A.X staining seen in mitotic, but not interphase, cells suggests that this aberrant mitosis results in DNA damage.

Mammals divert endogenous genotoxic formaldehyde into one-carbon metabolism

The folate-driven one-carbon (1C) cycle is a fundamental metabolic hub in cells that enables the synthesis of nucleotides and amino acids and epigenetic modifications. This cycle might also release formaldehyde, a potent protein and DNA crosslinking agent that organisms produce in substantial quantities. Here we show that supplementation with tetrahydrofolate, the essential cofactor of this cycle, and other oxidation-prone folate derivatives kills human, mouse and chicken cells that cannot detoxify formaldehyde or that lack DNA crosslink repair. Notably, formaldehyde is generated from oxidative decomposition of the folate backbone. Furthermore, we find that formaldehyde detoxification in human cells generates formate, and thereby promotes nucleotide synthesis. This supply of 1C units is sufficient to sustain the growth of cells that are unable to use serine, which is the predominant source of 1C units. These findings identify an unexpected source of formaldehyde and, more generally, indicate that the detoxification of this ubiquitous endogenous genotoxin creates a benign 1C unit that can sustain essential metabolism.

Real-time multi-marker measurement of organic compounds in human breath : towards fingerprinting breath.

The prospects for exploiting proton transfer reaction-time of flight-mass spectrometry (PTR-ToF-MS) in medical diagnostics are illustrated through a series of case studies. Measurements of acetone levels in the breath of 68 healthy people are presented along with a longitudinal study of a single person over a period of 1 month. The median acetone concentration across the population was 484 ppbV with a geometric standard deviation (GSD) of 1.6, whilst the average GSD during the single subject longtitudinal study was 1.5. An additional case study is presented which highlights the potential of PTR-ToF-MS in pharmacokinetic studies, based upon the analysis of online breath samples of a person following the consumption of ethanol. PTR-ToF-MS comes into its own when information across a wide mass range is required, particularly when such information must be gathered in a short time during a breathing cycle. To illustrate this property, multicomponent breath analysis in a small study of cystic fibrosis patients is detailed, which provides tentative evidence that online PTR-ToF-MS analysis of tidal breath can distinguish between active infection and non-infected patients.

Quantitation of salbutamol using micro-volume blood sampling - applications to exacerbations of pediatric asthma.

ABSTRACT Objectives: A novel gas chromatography-mass spectrometry (GC-MS) method has been developed to quantify salbutamol in micro-volumes (10 µL) of blood. A potential application is paediatric therapeutic dose monitoring (TDM) in acute severe asthma. Methods: At presentation, the children receive multiple doses of salbutamol (inhaled, nebulised and occasionally intravenous) but it is difficult to distinguish children who do not respond to treatment because of inadequate concentrations from those with toxicity, as symptoms are similar. A comparison was made between traditional dried blood spots (DBS) and the newly developed technique volumetric absorptive micro-sampling (VAMS), with specific investigation into the effect of drying time on analyte recovery. Results: For both sampling techniques, the final assay demonstrated good precision and accuracy across the concentration range tested (3–100 ng/mL), including both the normal therapeutic and toxic range. The method was developed to comply with FDA guidelines with precision and accuracy ≤15% for all concentrations, except the limit of quantification (5 ng/mL) where they were ≤20%. VAMS offered advantages in sampling ease and reduced GC-MS interference. The assay was successfully applied to the quantification of blood salbutamol concentrations in three healthy volunteers dosed with 1 mg salbutamol by inhalation. Conclusions: This demonstrated its potential for use in paediatric TDM studies, where in the acute situation considerably higher doses of salbutamol will have been administered. This is the first time that a TDM method for salbutamol has been carried out using VAMS and offers all the advantages provided by DBS, whilst eliminating the inherent sampling volume inaccuracies of traditional DBS collection.