John Turton

Investigations into the effects of various hepatotoxic compounds on urinary and liver taurine levels in rats

The effect of various hepatotoxicants on urinary taurine and urinary creatine has been studied in the rat. Several hepatotoxic agents, carbon tetrachloride, thioacetamide, galactosamine and allyl alcohol which all caused hepatic necrosis (sometimes accompanied by steatosis), resulted in a rise in urinary taurine and in some cases creatine, when administered to rats. Ethionine and hydrazine also raised urinary taurine but caused only steatosis and did not raise urinary creatine. Therefore urinary taurine and possibly creatine may be useful markers of liver injury and dysfunction. Liver taurine levels were also affected by some of the hepatotoxicants but in those cases where there was a rise in urinary taurine this could not be accounted for by the loss in liver taurine. It is suggested that the increase in urinary taurine is partly due to changes in protein synthesis and hence in sulphur amino acid metabolism caused by hepatotoxic agents. However, bromobenzene did not increase urinary taurine andα-naphthylisothiocyanate and lithocholate caused reduced levels. It is suggested that this lack of increase in urinary taurine may be due to depletion of glutathione or interference with the biliary system.

Liquid chromatography-mass spectrometry utilizing multi-stage fragmentation for the identification of oxysterols

In humans, the brain accounts for about 20% of the bodys free cholesterol, most of which is synthesized de novo in brain. To maintain cholesterol balance throughout life, cholesterol becomes metabolized to 24S-hydroxycholesterol, principally in neurons. In mouse, rat, and probably human, metabolism to 24S-hydroxycholesterol accounts for about 50% of cholesterol turnover; however, the route by which the remainder is turned over has yet to be elucidated. Here, we describe a novel liquid chromatography (LC) multi-stage fragmentation mass spectrometry (MSn) methodology for the identification, with high sensitivity (low pg), of cholesterol metabolites in rat brain. The methodology includes derivatization to enhance ionization, exact mass analysis at high resolution to identify potential metabolites, and LC-MSn (n=3) to allow their characterization. 24S-hydroxycholesterol was confirmed as a major oxysterol in rat brain, and other oxysterols identified for the first time in brain included 24,25-, 24,27-, 25,27-, 6,24,- 7α,25-, and 7α,27-dihydroxycholesterols. In addition, 3β-hydroxy-5-oxo-5,6-secocholestan-6-al and its aldol, two molecules linked to amyloidogenesis of proteins, were characterized in rat brain.

Distribution, Metabolism and Tumoricidal Activity of Doxorubicin Administered in Sorbitan Monostearate (Span 60) Niosomes in the Mouse

AbstractPurpose. Encapsulation of doxorubicin in niosomes was sought as a route to tumour targeting and improved tumoricidal through the alteration of doxorubicin pharmacokinetics and metabolism. Methods. Doxorubicin niosomes (10 mg kg−l doxorubicin) prepared from sorbitan monostearate (Span 60), cholesterol and choleth-24 (a 24 oxyethylene cholesteryl ether) in the molar ratio 45:45:10 were administered intravenously to female NMRI mice bearing the MAC 15A subcutaneously implanted tumour. Plasma doxorubicin was fractionated by gel filtration and quantified by HPLC with fluorometric detection as niosome-associated doxorubicin and released doxorubicin. Tumoricidal activity of the formulation was assessed by the intravenous injection of 5 mg kg−1 and 10 mg kg−1 doxorubicin niosomes to male NMRI mice bearing a 6 day old MAC 15A tumour. Results. At least 90% of the plasma doxorubicin was associated with the niosome fraction 4 h after dosing, and 50% was still associated after 24 h. The clearance of doxorubicin released from the niosomes was about 10 fold greater than the clearance of niosomal doxorubicin (176.5 mL h−l and 16.2 mL h−1, respectively). The area under the plasma level-time curve increased 6 fold when doxorubicin was administered in niosomes, compared to doxorubicin solution (66.0 µg.h mL−l and 10.3 µg.h mL−1, respectively). The area under the tumour level time curve was increased by over 50% by the administration of doxorubicin in niosomes when compared to the drug administered in solution (58.6 µg.h mL−l and 34.3 µg.h mL−1, respectively). There was no statistically significant difference between levels of the drug in the heart when niosomal doxorubicin or doxorubicin solution were administered. Doxorubicin metabolites, namely doxorubicinol and the aglycones doxorubicinone, doxorubicinolone and 7-deoxydoxorubicinone, were found associated with the niosomes in the plasma, possibly due to their adsorption to the vesicle surface once formed outside the niosome. Overall metabolite levels in the liver were increased when doxorubicin niosomes were administered compared to the drug in solution. A 5 mg kg−1 injection of doxorubicin niosomes produced a terminal mean tumour weight that was similar to that obtained from animals administered 10 mg kg−1 doxorubicin solution. Conclusions. Modest tumour targeting was achieved by the delivery of doxorubicin in sorbitan monostearate niosomes, increasing the tumour to heart AUC0–24 ratio from 0.27 to 0.36 and a doubling of tumoricidal activity. The overall level of doxorubicin metabolites was also increased.

Characterization of troponin responses in isoproterenol-induced cardiac injury in the Hanover Wistar rat.

The investigations aimed to evaluate the usefulness of cardiac troponins as biomarkers of acute myocardial injury in the rat. Serum from female Hanover Wistar rats treated with a single intraperitoneal (IP) injection of isoproterenol (ISO) was assayed for cardiac troponin I (cTnI) (ACS: 180SE, Bayer), cTnI (Immulite 2000, Diagnostic Products Corporation) and cardiac troponin T (cTnT) (Elecsys 2010, Roche). In a time-course study (50.0 mg/kg ISO), serum cTnI (ACS:180SE) and cTnT increased above control levels at 1 hour postdosing, peaking at 2 hours (cTnI, 4.30 μg/L; cTnT, 1.79 μg/L), and declined to baseline by 48 hours, with histologic cardiac lesions first seen at 4 hours postdosing. The Immulite 2000 assay gave minimal cTnI signals, indicating poor immunoreactivity towards rat cTnI. In a dose-response study (0.25 to 20.0 mg/kg ISO), there was a trend for increasing cTnI (ACS:180SE) values with increasing ISO dose levels at 2 hours postdosing. By 24 hours, cTnI levels returned to baseline although chronic cardiac myodegeneration was present. We conclude that serum cTnI and cTnT levels are sensitive and specific biomarkers for detecting ISO induced myocardial injury in the rat. Serum troponin values reflect the development of histopathologic lesions; however peak troponin levels precede maximal lesion severity.

Application of surface-enhanced laser desorption/ionization technology to the detection and identification of urinary parvalbumin-α : a biomarker of compound-induced skeletal muscle toxicity in the rat

In toxicity studies, compound‐induced changes are typically evaluated using a combination of endpoints and there are often a number of potential markers in biological fluids which can indicate toxic change in tissues and organs. However, some biomarkers are not specific to the organ of injury and therefore there is a continuing search for more sensitive and specific indicators of target organ toxicity. In experiments to assess the potential diagnostic usefulness of surface‐enhanced laser desorption/ionization (SELDI) ProteinChip® technology, skeletal muscle toxicity was induced in Wistar Han rats by administering 2,3,5,6‐tetramethyl‐p‐phenylenediamine (TMPD). The skeletal muscle toxicity was monitored using established endpoints such as increase in serum aldolase (Aldol), aspartate aminotransferase (AST) and histopathology, and also using SELDI retentate chromatography mass spectrometry of urine samples. Clear differences in urinary protein patterns between control and TMPD‐treated animals were observed on the ProteinChip surfaces. Additionally a specific urine marker protein of 11.8 kDa was identified in TMPD‐dosed rats, and the detection of the marker was related to the degree of skeletal muscle toxicity assessed by recognized clinical pathology endpoints. The 11.8 kDa protein was identified as parvalbumin‐α. These experiments demonstrated the potential of urinary parvalbumin‐α as a specific, noninvasive, and easily detectable biomarker for skeletal muscle toxicity in the rat and the potential of SELDI technology for biomarker detection and identification in toxicology studies.

Time Course Characterization of Serum Cardiac Troponins, Heart Fatty Acid-binding Protein, and Morphologic Findings With Isoproterenol-induced Myocardial Injury in the Rat

We investigated the kinetics of circulating biomarker elevation, specifically correlated with morphology in acute myocardial injury. Male Hanover Wistar rats underwent biomarker and morphologic cardiac evaluation at 0.5 to seventy-two hours after a single subcutaneous isoproterenol administration (100 or 4000 µg/kg). Dose-dependent elevations of serum cardiac troponins I and T (cTnI, cTnT), and heart fatty acid–binding protein (H-FABP) occurred from 0.5 hour, peaked at two to three hours, and declined to baseline by twelve hours (H-FABP) or forty-eight to seventy-two hours (Serum cTns). They were more sensitive in detecting cardiomyocyte damage than other serum biomarkers. The Access 2 platform, an automated chemiluminescence analyzer (Beckman Coulter), showed the greatest cTnI fold-changes and low range sensitivity. Myocardial injury was detected morphologically from 0.5 hour, correlating well with loss of cTnI immunoreactivity and serum biomarker elevation at early time points. Ultrastructurally, there was no evidence of cardiomyocyte death at 0.5 hour. After three hours, a clear temporal disconnect occurred: lesion scores increased with declining cTnI, cTnT, and H-FABP values. Serum cTns are sensitive and specific markers for detecting acute/active cardiomyocyte injury in this rat model. Heart fatty acid–binding protein is a good early marker but is less sensitive and nonspecific. Release of these biomarkers begins early in myocardial injury, prior to necrosis. Assessment of cTn merits increased consideration for routine screening of acute/ongoing cardiomyocyte injury in rat toxicity studies.

Systemic antiangiogenic activity of cationic poly-L-lysine dendrimer delays tumor growth

This study describes the previously unreported intrinsic capacity of poly-L-lysine (PLL) sixth generation (G6) dendrimer molecules to exhibit systemic antiangiogenic activity that could lead to solid tumor growth arrest. The PLL-dendrimer-inhibited tubule formation of SVEC4-10 murine endothelial cells and neovascularization in the chick embryo chick chorioallantoic membrane (CAM) assay. Intravenous administration of the PLL-dendrimer molecules into C57BL/6 mice inhibited vascularisation in Matrigel plugs implanted subcutaneously. Antiangiogenic activity was further evidenced using intravital microscopy of tumors grown within dorsal skinfold window chambers. Reduced vascularization of P22 rat sarcoma implanted in the dorsal window chamber of SCID mice was observed following tail vein administration (i.v.) of the PLL dendrimers. Also, the in vivo toxicological profile of the PLL-dendrimer molecules was shown to be safe at the dose regime studied. The antiangiogenic activity of the PLL dendrimer was further shown to be associated with significant suppression of B16F10 solid tumor volume and delayed tumor growth. Enhanced apoptosis/necrosis within tumors of PLL-dendrimer-treated animals only and reduction in the number of CD31 positive cells were observed in comparison to protamine treatment. This study suggests that PLL-dendrimer molecules can exhibit a systemic antiangiogenic activity that may be used for therapy of solid tumors, and in combination with their capacity to carry other therapeutic or diagnostic agents may potentially offer capabilities for the design of theranostic systems.

Reduction of liver taurine in rats by β-alanine treatment increases carbon tetrachloride toxicity

Treatment of rats with beta-alanine increases the urinary taurine levels and markedly reduces the concentration of taurine in the liver. Dosing with carbon tetrachloride (CCl4) during treatment with beta-alanine results in a marked decrease in urinary taurine concomitant with a decrease in food intake. Treatment of animals with beta-alanine increases the hepatotoxicity of single doses of CCl4 as determined histologically and by measurement of serum alanine transaminase (ALT) and aspartate transaminase (AST) levels. Urinary creatine is also raised significantly after the administration of CCl4 in beta-alanine-treated animals. However, the accumulation of triglycerides (TRIG) in the liver caused by dosing with CCl4 was not influenced by beta-alanine treatment. The data suggest that liver taurine levels may be an important factor in determining the degree of CCl4-induced cellular necrosis but not hepatic triglyceride accumulation.

Taurine, a possible urinary marker of liver damage: a study of taurine excretion in carbon tetrachloride-treated rats

Carbon tetrachloride (CCl4) caused a dose-dependent increase in urinary taurine which correlated with both the histological and biochemical assessment of liver damage. The peak elevation in urinary taurine occurred within the first 48 h after dosing but there was still significant taurinuria 72 and 96 h after the intermediate dose (1 ml.kg−1) and highest dose (2 ml.kg−1), respectively. Levels of taurine in serum were also elevated over the 24 h period following a hepatotoxic dose (2 ml.kg−1) of CCl4. In contrast, although initially elevated, levels of taurine in the liver declined over the 24 h period following dosing and were significantly lower 96 h after a hepatotoxic dose of CCl4 (2 ml.kg−1). Male rats showed a different urinary profile for taurine than female rats after dosing with CCl4. A reduction in food intake seemed to lower urinary taurine levels although these changes were not statistically significant. There was a significant correlation between the level of urinary taurine and the level of serum AST for individual animals given a hepatotoxic dose of CCl4 (2 ml.kg−1). The data presented suggest that: i) taurine is produced by the liver in response to a toxic insult and subsequent leakage from damaged cells leads to increased levels in the urine; ii) the urinary taurine level may be a useful non-invasive marker of liver damage.

The calcium channel blocker lacidipine reduces the development of atherosclerotic lesions in the apoE-deficient mouse.

Background Lacidipine is a widely used calcium-channel blocker, which has both long-lasting antihypertensive activity and also antioxidant properties. Previous studies have demonstrated the ability of lacidipine to reduce the development of atherosclerotic lesions in several animal models. Objective The present study investigated the anti-atherosclerotic potential of lacidipine in the apoE-deficient mouse, an experimental model of atherosclerosis showing progressively complex and widespread lesions which closely resemble the inflammatory-fibrous plaques seen in humans. Methods Lacidipine was administered daily by gavage for 10 weeks at dose levels of 0 (control), 0.3, 1.0 and 3.0 mg/kg. Results Lacidipine administration reduces the extension of atherosclerotic lesions in the aorta of the apoE-deficient mouse without affecting plasma lipid levels. We also show that apoE-deficient mice have four-fold higher values of the proatherogenic peptide, endothelin, compared with the wild-type C57BL/6 mouse and that lacidipine administration reduced, in a dose-dependent manner, the concentrations of plasma endothelin. Conclusion Lacidipine has anti-atherogenic effects in the apoE-deficient mouse, and reduces plasma endothelin concentrations.