Pharmacology and Therapeutics - Research Publications

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    Iron overload and impaired iron handling contribute to the dystrophic pathology in models of Duchenne muscular dystrophy
    Alves, FM ; Kysenius, K ; Caldow, MK ; Hardee, JP ; Chung, JD ; Trieu, J ; Hare, DJ ; Crouch, PJ ; Ayton, S ; Bush, A ; Lynch, GS ; Koopman, R (WILEY, 2022-03-06)
    BACKGROUND: Oxidative stress is implicated in the pathophysiology of Duchenne muscular dystrophy (DMD, caused by mutations in the dystrophin gene), which is the most common and severe of the muscular dystrophies. To our knowledge, the distribution of iron, an important modulator of oxidative stress, has not been assessed in DMD. We tested the hypotheses that iron accumulation occurs in mouse models of DMD and that modulation of iron through the diet or chelation could modify disease severity. METHODS: We assessed iron distribution and total elemental iron using LA-ICP-MS on skeletal muscle cross-sections of 8-week-old Bl10 control mice and dystrophic mdx mice (with moderate dystrophy) and dystrophin/utrophin-null mice (dko, with severe dystrophy). In addition, mdx mice (4 weeks) were treated with either an iron chelator (deferiprone 150 mg/kg/day) or iron-enriched feed (containing 1% added iron as carbonyl iron). Immunoblotting was used to determine the abundance of iron- and mitochondria-related proteins. (Immuno)histochemical and mRNA assessments of fibrosis and inflammation were also performed. RESULTS: We observed a significant increase in total elemental iron in hindlimb muscles of dko mice (+50%, P < 0.05) and in the diaphragm of mdx mice (+80%, P < 0.05), with both tissues exhibiting severe pathology. Iron dyshomeostasis was further evidenced by an increase in the storage protein ferritin (dko: +39%, P < 0.05) and ferroportin compared with Bl10 control mice (mdx: +152% and dko: +175%, P < 0.05). Despite having features of iron overload, dystrophic muscles had lower protein expression of ALAS-1, the rate-limiting enzyme for haem synthesis (dko -44%, P < 0.05), and the haem-containing protein myoglobin (dko -54%, P < 0.05). Deferiprone treatment tended to decrease muscle iron levels in mdx mice (-30%, P < 0.1), which was associated with lower oxidative stress and fibrosis, but suppressed haem-containing proteins and mitochondrial content. Increasing iron via dietary intervention elevated total muscle iron (+25%, P < 0.05) but did not aggravate the pathology. CONCLUSIONS: Muscles from dystrophic mice have increased iron levels and dysregulated iron-related proteins that are associated with dystrophic pathology. Muscle iron levels were manipulated by iron chelation and iron enriched feed. Iron chelation reduced fibrosis and reactive oxygen species (ROS) but also suppressed haem-containing proteins and mitochondrial activity. Conversely, iron supplementation increased ferritin and haem-containing proteins but did not alter ROS, fibrosis, or mitochondrial activity. Further studies are required to investigate the contribution of impaired ferritin breakdown in the dysregulation of iron homeostasis in DMD.
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    An integrated mass spectrometry imaging and digital pathology workflow for objective detection of colorectal tumours by unique atomic signatures
    Paul, B ; Kysenius, K ; Hilton, JB ; Jones, MWM ; Hutchinson, RW ; Buchanan, DD ; Rosty, C ; Fryer, F ; Bush, A ; Hergt, JM ; Woodhead, JD ; Bishop, DP ; Doble, PA ; Hill, MM ; Crouch, PJ ; Hare, DJ (ROYAL SOC CHEMISTRY, 2021-06-29)
    Tumours are abnormal growths of cells that reproduce by redirecting essential nutrients and resources from surrounding tissue. Changes to cell metabolism that trigger the growth of tumours are reflected in subtle differences between the chemical composition of healthy and malignant cells. We used LA-ICP-MS imaging to investigate whether these chemical differences can be used to spatially identify tumours and support detection of primary colorectal tumours in anatomical pathology. First, we generated quantitative LA-ICP-MS images of three colorectal surgical resections with case-matched normal intestinal wall tissue and used this data in a Monte Carlo optimisation experiment to develop an algorithm that can classify pixels as tumour positive or negative. Blinded testing and interrogation of LA-ICP-MS images with micrographs of haematoxylin and eosin stained and Ki67 immunolabelled sections revealed Monte Carlo optimisation accurately identified primary tumour cells, as well as returning false positive pixels in areas of high cell proliferation. We analysed an additional 11 surgical resections of primary colorectal tumours and re-developed our image processing method to include a random forest regression machine learning model to correctly identify heterogenous tumours and exclude false positive pixels in images of non-malignant tissue. Our final model used over 1.6 billion calculations to correctly discern healthy cells from various types and stages of invasive colorectal tumours. The imaging mass spectrometry and data analysis methods described, developed in partnership with clinical cancer researchers, have the potential to further support cancer detection as part of a comprehensive digital pathology approach to cancer care through validation of a new chemical biomarker of tumour cells.
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    Superoxide Dismutase 1 in Health and Disease: How a Frontline Antioxidant Becomes Neurotoxic
    Trist, BG ; Hilton, JB ; Hare, DJ ; Crouch, PJ ; Double, KL (WILEY-V C H VERLAG GMBH, 2020-11-19)
    Cu/Zn superoxide dismutase (SOD1) is a frontline antioxidant enzyme catalysing superoxide breakdown and is important for most forms of eukaryotic life. The evolution of aerobic respiration by mitochondria increased cellular production of superoxide, resulting in an increased reliance upon SOD1. Consistent with the importance of SOD1 for cellular health, many human diseases of the central nervous system involve perturbations in SOD1 biology. But far from providing a simple demonstration of how disease arises from SOD1 loss-of-function, attempts to elucidate pathways by which atypical SOD1 biology leads to neurodegeneration have revealed unexpectedly complex molecular characteristics delineating healthy, functional SOD1 protein from that which likely contributes to central nervous system disease. This review summarises current understanding of SOD1 biology from SOD1 genetics through to protein function and stability.