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Dive into the research topics where Mark Graham is active.

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Featured researches published by Mark Graham.


Toxicology | 2008

Induction of drug metabolism: Species differences and toxicological relevance

Mark Graham; Brian G. Lake

A large number of drugs and other chemicals have been shown to induce hepatic microsomal cytochrome P450 (CYP) forms in experimental animals and humans. Most CYP forms are induced by receptor-mediated mechanisms leading to an increase in gene transcription. Important nuclear receptors involved in the induction of CYP1A, CYP2B, CYP3A and CYP4A subfamily forms comprise, respectively, the aryl hydrocarbon receptor, the constitutive androstane receptor, the pregnane X receptor and the peroxisome proliferator-activated receptor alpha. Hepatic CYP form induction can be assessed by in vivo, ex vivo and in vitro methods. Significant species differences can exist in the enzyme induction response to a given chemical and also in the toxicological consequences of induction. Hepatic CYP form induction in humans may lead to clinically important drug-drug interactions. In rodents hepatic CYP form induction can be associated with the formation of tumours by non-genotoxic modes of action in the liver, thyroid and other tissues.


Mutagenesis | 2008

Cellular protection from oxidative DNA damage by over-expression of the novel globin cytoglobin in vitro

Nikolos J. Hodges; Neal Innocent; Subdha Dhanda; Mark Graham

Cytoglobin is a recently identified member of the mammalian globin family that is expressed in neuronal cells in the central and peripheral nervous system where its physiological role remains to be determined. In the current study, we demonstrate that a cytoglobin-green fluoresecent protein (GFP) fusion protein when expressed in the human neuronal cell line TE671 has a nuclear localization in a subpopulation of transfected cells (approximately 15%). Furthermore, the cytoglobin-GFP fusion protein but not GFP alone significantly reduced the induction of intracellular reactive oxygen species as assessed by oxidation of the redox-sensitive probe dichlorofluorescein following treatment with non-cytotoxic concentrations of the pro-oxidant Ro19-8022. In addition, expression of cytoglobin-GFP also afforded cytoprotection from Ro19-8022-induced oxidative DNA damage as assessed by the Fpg-modified comet assay. In conclusion, the current study provides evidence supportive of a role for cytoglobin in cytoprotection of neuronal cells from oxidative-related damage, for example, during ischaemic reperfusion injury following hypoxia.


Xenobiotica | 2003

mRNA and protein expression of dog liver cytochromes P450 in relation to the metabolism of human CYP2C substrates

Mark Graham; A. R. Bell; H. K. Crewe; C. L. Moorcraft; L. Walker; E. F. Whittaker; M. S. Lennard

1. Interpretation of novel drug exposure and toxicology data from the dog is tempered by our limited molecular and functional knowledge of dog cytochromes P450 (CYPs). The aim was to study the mRNA and protein expression of hepatic dog CYPs in relation to the metabolism of substrates of human CYP, particularly those of the CYP2C subfamily. 2. The rate of 7-hydroxylation of S -warfarin (CYP2C9 in humans) by dog liver microsomes (mean ± SD from 12 (six male and six female) dogs = 10.8 ± 1.9 fmolu2009mg − 1 proteinu2009min − 1) was 1.5-2 orders of magnitude lower than that in humans. 3. The rate of 4-hydroxylation of S -mephenytoin, catalysed in humans by CYP2C19, was also low in dog liver (4.6 ± 1.5 pmolu2009mg − 1 proteinu2009min − 1) compared with human liver. In contrast, the rate of 4-hydroxylation of the R -enantiomer of mephenytoin by dog liver was much higher. The kinetics of this reaction (range of K m or K 0.5 15-22 µM, V max 35-59 pmolu2009mg − 1 proteinu2009min − 1, n = 4 livers) were consistent with the involvement of a single enzyme. 4. In contrast to our findings for S -mephenytoin, dog liver microsomes 5-hydroxylated omeprazole (also catalysed by CYP2C19 in humans) at considerably higher rates (range of K m 42-64 µM, V max 22-46 pmolu2009mg − 1 proteinu2009min − 1, n = 4 livers). 5. For all the substrates except omeprazole, a sex difference in their metabolism was observed in the dog (dextromethorphan N-demethylation: female range = 0.7-0.9, male = 0.4-0.8 nmolu2009mg − 1 proteinu2009min − 1 (p < 0.02); S -warfarin 7-hydroxylation: female = 9-15.5, male = 8-12 fmolu2009mg − 1 proteinu2009min − 1 (p < 0.02); R -mephenytoin 4-hydroxylation: female = 16-35, male = 11.5-19 pmolu2009mg − 1 proteinu2009min − 1 (p < 0.01); omeprazole 5-hydroxylation: female = 15-20, male 13-22 pmolu2009mg − 1 proteinu2009min − 1 (p < 0.2)). 6. All dog livers expressed mRNA and CYP3A12, CYP2B11, CYP2C21 proteins, with no sex differences being found. Expression of CYP2C41 mRNA was undetectable in the livers of six of 11 dogs. 7. Correlation analysis suggested that CYP2B11 catalyses the N-demethylation of dextromethorphan (mediated in humans by CYP3A) and the 4-hydroxylation of mephenytoin (mediated in humans by CYP2C19) in the dog, and that this enzyme and CYP3A12 contribute to S -warfarin 7-hydroxylation (mediated in humans by CYP2C9). 8. In conclusion, we have identified a distinct pattern of hepatic expression of the CYP2C41 gene in the Alderley Park beagle dog. Furthermore, marked differences in the metabolism of human CYP2C substrates were observed in this dog strain compared with humans with respect to rate of reaction, stereoselectivity and CYP enzyme selectivity.


Xenobiotica | 2008

Nuclear hormone receptor-dependent regulation of hepatic transporters and their role in the adaptive response in cholestasis

S. Stahl; M. R. Davies; D. I. Cook; Mark Graham

1.u2003Hepatobiliary transport systems are essential for the uptake and excretion of a variety of organic anions including bile acids and bilirubin. Perturbation of this vital liver function can result in pathological conditions such as cholestasis, where the formation of bile at the canaliculus is impaired resulting in the intrahepatic accumulation of toxic bile constituents. 2.u2003Members of the nuclear hormone receptor superfamily are important mediators of the adaptive response during cholestasis controlling the expression of transporters and other proteins with the aim to limit tissue damage. Bile acids are the endogenous ligands for these nuclear hormone receptors and therefore directly participate in the control of their own transport and metabolism. 3.u2003Adaptive events include repression of bile acid uptake and de novo bile acid synthesis as well as a concomitant induction of alternative efflux routes and bile acid detoxification. Importantly, the adaptation also extends to other organs such as intestine and kidney to facilitate elimination of bile acids from the body. 4.u2003This review provides an overview of the transcriptional regulation of bile acid transporting proteins and metabolizing enzymes mediated by nuclear hormone receptors. Furthermore, the complex networks between nuclear hormone receptors and regulated genes are illustrated and implications of targeting these receptors for the treatment of cholestasis are discussed.


Mutagenesis | 2008

Reactive oxygen species from the uncoupling of human cytochrome P450 1B1 may contribute to the carcinogenicity of dioxin-like polychlorinated biphenyls

Richard M. Green; Nikolas J. Hodges; J. Kevin Chipman; Michael R. O'Donovan; Mark Graham

Polychlorinated biphenyls (PCBs) are classified by the International Agency for Research on Cancer as probable human carcinogens. A subset of PCBs are described as dioxin like because of similarities to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Dioxin-like PCBs have been shown to tightly bind the active site of cytochrome P450 (CYP) 1A isoforms, primarily CYP1A1, resulting in inhibition of CYP activity and the generation of reactive oxygen species (ROS) as a result of uncoupling of the catalytic cycle. Human CYP1B1 (hCYP1B1) is an extrahepatic CYP closely related to hCYP1A1 and is overexpressed in the lungs of smokers. Moreover, hCYP1B1 has been found to be overexpressed in cancers derived from a number of tissue types, as well as in pre-malignant prostate tumours, implicating overexpression of hCYP1B1 as a risk factor for extrahepatic carcinogenesis. It has been demonstrated previously that hCYP1B1 is inhibited by dioxin-like PCBs, but whether or not it is uncoupled has not been investigated. In the current study, the ability of three dioxin-like PCBs 3,3,4,4-tetrachlorobiphenyl, 3,3,4,4,5-pentachlorobiphenyl and 3,3,4,4,5,5-hexachlorobiphenyl (PCB169) to inhibit hCYP1B1 and stimulate the formation of ROS in V79MZ cells (which lack endogenous CYPs) expressing hCYP1B1 was demonstrated. Moreover, the generation of ROS was also associated with increases in parameters of oxidative stress related to genotoxicity (DNA oxidation and lipid peroxidation). For PCB169, these effects were time and concentration dependent. These data identify a novel mechanism of genotoxicity for dioxin-like PCBs, as well as providing further evidence that overexpression of hCYP1B1 is a risk factor for extrahepatic carcinogenesis.


Brain Research | 2003

The monocarboxylate transport inhibitor, α-cyano-4-hydroxycinnamate, has no effect on retinal ischemia

José Melena; Rukhsana Safa; Mark Graham; Robert J. Casson; Neville N. Osborne

Glial-derived monocarboxylate lactate is thought to be an important energy source for neurons during brain activation or in hypoxia-ischemia. Treatment with alpha-cyano-4-hydroxycinnamate (4-CIN), a monocarboxylate transporter inhibitor, has been recently reported to exacerbate delayed neuronal damage in a rat model of cerebral ischemia, an effect ascribed to inhibition of lactate/pyruvate transport. Since monocarboxylate transporters are abundant in the retina, we examined the effect of 4-CIN administration on the outcome of high intraocular pressure-induced retinal ischemia in rats. Retinal ischemic damage was assessed by changes in the electroretinogram (ERG), the retinal localization of choline acetyltransferase (ChAT) and neuronal nitric oxide synthase (nNOS) immunoreactivities, and the loss of retinal mRNA for Thy-1. Intraperitoneal or intravitreal administration of 4-CIN had no effect on the ERG or the localization of ChAT and nNOS immunoreactivities in either the control retina or a retina subjected to ischemia/reperfusion. In addition, intravitreal injection of 4-CIN had no effect on ischemia-induced reduction of retinal mRNA levels for Thy-1. These results provide no evidence to support the view that blockade of lactate uptake and/or pyruvate entry into mitochondria for oxidative metabolism has an influence on the outcome of retinal ischemia/reperfusion.


Toxicology and Applied Pharmacology | 2003

Lansoprazole increases testosterone metabolism and clearance in male Sprague-Dawley rats: implications for Leydig cell carcinogenesis

Michelle Coulson; G. Gordon Gibson; Nick Plant; Timothy G. Hammond; Mark Graham

Leydig cell tumours (LCTs) are frequently observed during rodent carcinogenicity studies, however, the significance of this effect to humans remains a matter of debate. Many chemicals that produce LCTs also induce hepatic cytochromes P450 (CYPs), but it is unknown whether these two phenomena are causally related. Our aim was to investigate the existence of a liver-testis axis wherein microsomal enzyme inducers enhance testosterone metabolic clearance, resulting in a drop in circulating hormone levels and a consequent hypertrophic response from the hypothalamic-pituitary-testis axis. Lansoprazole was selected as the model compound as it induces hepatic CYPs and produces LCTs in rats. Male Sprague-Dawley rats were dosed with lansoprazole (150 mg/kg/day) or vehicle for 14 days. Lansoprazole treatment produced effects on the liver consistent with an enhanced metabolic capacity, including significant increases in relative liver weights, total microsomal CYP content, individual CYP protein levels, and enhanced CYP-dependent testosterone metabolism in vitro. Following intravenous administration of [14C]testosterone, lansoprazole-treated rats exhibited a significantly smaller area under the curve and significantly higher plasma clearance. Significant reductions in plasma and testicular testosterone levels were observed, confirming the ability of this compound to perturb androgen homeostasis. No significant changes in plasma LH, FSH, or prolactin levels were detected under our experimental conditions. Lansoprazole treatment exerted no marked effects on testicular testosterone metabolism. In summary, lansoprazole treatment induced hepatic CYP-dependent testosterone metabolism in vitro and enhanced plasma clearance of radiolabelled testosterone in vivo. These effects may contribute to depletion of circulating testosterone levels and hence play a role in the mode of LCT induction in lansoprazole-treated rats.


Fibrogenesis & Tissue Repair | 2015

Cytoglobin expression in the hepatic stellate cell line HSC-T6 is regulated by extracellular matrix proteins dependent on FAK-signalling

Louise Catherine Stone; Lorna Susan Thorne; Chris J. Weston; Mark Graham; Nikolas J. Hodges

BackgroundFibrosis is a physiological response to cellular injury in the liver and is mediated by the activation of hepatic stellate cells resulting in the replacement of hepatocytes with extracellular matrix comprised principally of collagen 1 to form a hepatic scar. Although the novel hexaco-ordinated globin cytoglobin was identified in activated hepatic stellate cells more than 10xa0years ago, its role in stellate cell biology and liver fibrosis remains enigmatic.ResultsIn the current study, we investigated the role of different extracellular matrix proteins in stellate cell proliferation, activation (alpha smooth muscle actin expression and retinoic acid uptake) and cytoglobin expression. Our results demonstrate that cytoglobin expression is correlated with a more quiescent phenotype of stellate cells in culture and that cytoglobin is regulated by the extracellular matrix through integrin signalling dependent on activation of focal adhesion kinase.ConclusionsAlthough further studies are required, we provide evidence that cytoglobin is a negative regulator of stellate cell activation and therefore may represent a novel target for anti-fibrotic treatments in the future.


Xenobiotica | 2008

Quantitative analysis of aryl hydrocarbon receptor activation using fluorescence-based cell imaging—A high-throughput mechanism-based assay for drug discovery

Helen Garside; Allison Stewart; Nick Brown; Emma-Louise Cooke; Mark Graham; Michael Sullivan

Early identification of toxicity associated with new chemical entities is important for reducing compound attrition in late stage drug discovery. Activation of the aryl hydrocarbon receptor (AhR) by xenobiotics is a recognised mechanism of toxicity: the AhR mediates most, if not all, of the serious toxicities caused by the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In addition to compounds such as TCDD, the AhR can be activated by compounds with drug-like properties; consequently there is a desire to eliminate AhR activity in candidate drug programs. Endogenous AhR translocates from the cytoplasm to the nucleus in response to prototypical AhR ligands. This trafficking was monitored in mouse Hepa-1 cells, human HepG2 cells and rat primary hepatocytes using an anti-AhR antibody. A confocal imaging plate reader, the InCell® Analyzer 3000, was used to image fixed cells cultured in 96 well plates, and algorithms were used to analyse both population data and individual cell responses. The subsequent induction of the CYP1A1 gene, in the three cell models, was also assessed using quantitative real-time polymerase chain reaction and showed good correlation with the translocation assay. To conclude, we have established robust, automated high throughput assays for the identification of AhR activators in primary hepatocytes and cell lines.


Toxicological Sciences | 2017

From the Cover: Development and Application of a Dual Rat and Human AHR Activation Assay

Martin Brown; Helen Garside; Emma Thompson; Saseela Atwal; Chloe Bean; Tony Goodall; Michael Sullivan; Mark Graham

Significant prolonged aryl hydrocarbon receptor (AHR) activation, classically exhibited following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin, can cause a variety of undesirable toxicological effects. Novel pharmaceutical chemistries also have the potential to cause activation of AHR and consequent toxicities in pre-clinical species and man. Previous methods either employed relatively expensive and low-throughput primary hepatocyte dosing with PCR endpoint, or low resolution overexpressing reporter gene assays. We have developed, validated and applied an in vitro microtitre plate imaging-based medium throughput screening assay for the assessment of endogenous species-specific AHR activation potential via detection of induction of the surrogate transcriptional target Cytochrome P450 CYP1A1. Routine testing of pharmaceutical drug development candidate chemistries using this assay can influence the chemical design process and highlight AHR liabilities. This assay should be introduced such that human AHR activation liability is flagged early for confirmatory testing.

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