Catherine J. Waterfield

The in vivo and in vitro protective properties of taurine

1. Taurine is a ubiquitous, free amino acid found in mammalian systems. 2. The biological functions of taurine are unclear. 3. Various in vivo data suggest that taurine has a variety of protective functions and deficiency leads to pathological changes. 4. Depletion in rats of taurine increases susceptibility to liver damage from carbon tetrachloride. 5. Susceptibility to a variety of hepatotoxicants correlates with the estimated hepatic taurine level. 6. In vitro data suggest that taurine can protect cells against toxic damage. 7. Taurine protects isolated hepatocytes against carbon tetrachloride, hydrazine and 1,4-naphthoquinone but not against allyl alcohol, alpha-naphthylisothiocyanate (ANIT) or diaminodiphenyl methane (DAPM) cytotoxicity. 8. The mechanisms of protection are unclear but may include modulation of calcium levels, osmoregulation and membrane stabilization.

NMR and pattern recognition studies on liver extracts and intact livers from rats treated with α-naphthylisothiocyanate

The metabolite profiles from livers of toxin-treated rats were investigated using high resolution 1H NMR spectroscopy of aqueous (acetonitrile/water), lipidic (chloroform/methanol) extracts and magic angle spinning (MAS)-NMR spectroscopy of intact tissue. Rats were treated with the model cholestatic hepatotoxin, alpha-naphthylisothiocyanate (ANIT, 150 mg/kg) and NMR spectra of liver were analysed using principal components analysis (PCA) to extract novel toxicity biomarker information. 1H NMR spectra of control aqueous extracts showed signals from a range of organic acids and bases, amino acids, sugars, and glycogen. Chloroform/methanol extracts showed signals from a range of saturated and unsaturated triglycerides, phospholipids and cholesterol. The MAS 1H NMR spectra of livers showed a composite of signals found in both aqueous and lipophilic extracts. Following ANIT treatment, 1H NMR-PCA of aqueous extracts indicated a progressive reduction in glucose and glycogen, together with increases in bile acid, choline, and phosphocholine signals. 1H NMR-PCA of chloroform/methanol extracts showed elevated triglyceride levels. The 1H MAS-NMR-PCA analysis allowed direct detection of all of the ANIT-induced tissue perturbations revealed by 1H NMR of extracts, enabling metabolic characterisation of the lesion, which included steatosis, bile duct obstruction and altered glucose/glycogen metabolism. MAS-NMR spectroscopy requires minimal sample preparation and, unlike 1H NMR spectroscopy of tissue extracts, does not discriminate metabolites based on their solubility in a particular solvent and so this is a particularly useful exploratory tool in biochemical toxicology.

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.

Effects of feeding and body weight loss on the 1H-NMR-based urine metabolic profiles of male Wistar Han Rats: Implications for biomarker discovery

For almost two decades, 1H-NMR spectroscopy has been used as an ‘open’ system to study the temporal changes in the biochemical composition of biofluids, including urine, in response to adverse toxic events. Many of these in vivo studies have reported changes in individual metabolites and patterns of metabolites that correlated with toxicological changes. However, many of the proposed novel biomarkers are common to a number of different types of toxicity. These may therefore reflect non-specific effects of toxicity, such as weight loss, rather than a specific pathology. A study was carried out to investigate the non-specific effects on urinary metabolite profiles by administering four hepatotoxic compounds, as a single dose, to rats at two dose levels: hydrazine hydrate (0.06 or 0.08 g kg−1), 1,2-dimethylhydrazine (0.1 or 0.3 g kg−1), α-napthylisothiocyanate (0.1 or 0.15 g kg−1) and carbon tetrachloride (1.58 or 3.16 g kg−1). The study included weight-matched control animals along with those that were dosed, which were then ‘pair-fed’ with the treated animals so they achieved a similar weight loss. The urinary metabolite profiles were investigated over time using 1H-NMR spectroscopy and compared with the pathology from the same animals. The temporal changes were analysed statistically using multivariate statistical data analysis including principal component analysis, partial least squares, parallel factor analysis and Fishers criteria. A number of metabolites associated with energy metabolism or which are partially dietary in origin, such as creatine, creatinine, tricarboxylic acid (TCA) cycle intermediates, phenylacetylglycine, fumarate, glucose, taurine, fatty acids and N-methylnicotinamide, showed altered levels in the urine of treated and pair-fed animals. Many of these changes correlated well with weight loss. Interestingly, there was no increase in ketone bodies (acetate and β-hydroxybutyrate), which might be expected if energy metabolism was switched from glycolysis to fatty acid β-oxidation. In some instances, the metabolites that changed were considered to be non-specific markers of toxicity, but were also identified as markers of a specific type of toxicity. For example, taurine was raised significantly in carbon tetrachloride-treated animals but reduced in the pair-fed group. However, raised urinary bile acid levels were only seen after α-napthylisothiocyanate treatment. The methodology, statistical analysis used and the data generated will help improve the identification of specific markers or patterns of urinary markers of specific toxic effects.

A metabonomic approach to the investigation of drug-induced phospholipidosis: an NMR spectroscopy and pattern recognition study

1H NMR spectroscopy of urine and pattern recognition analysis have been used to study the metabolic perturbations caused following dosing of five novel drug candidates, two of which (GWA, GWB) caused mild lung and liver phospholipidosis, whilst the rest (GWC-GWE) did not cause any detectable toxicity. Urine samples were collected predose, 0-8 h, 8-16 h, 16-24 h and 24-32 h after single, oral dosing with each compound to Han Wistar rats (n = 3 per group), and liver and lung samples for were taken at 48 h for histology. 1H NMR spectra of whole urine were acquired, processed and subsequently analysed using principal component analysis. All animals administered the drug candidates showed a significant reduction in serum triglycerides and those animals administered either GWA or GWB were observed to have foamy alveolar macrophages and the presence of multilamellar bodies in hepatocytes by electron microscopy. In the plot of the first two principal components, urinary spectra of those animals dosed with GWA or ...1H NMR spectroscopy of urine and pattern recognition analysis have been used to study the metabolic perturbations caused following dosing of five novel drug candidates, two of which (GWA, GWB) caused mild lung and liver phospholipidosis, whilst the rest (GWC-GWE) did not cause any detectable toxicity. Urine samples were collected predose, 0-8 h, 8-16 h, 16-24 h and 24-32 h after single, oral dosing with each compound to Han Wistar rats (n = 3 per group), and liver and lung samples for were taken at 48 h for histology. 1H NMR spectra of whole urine were acquired, processed and subsequently analysed using principal component analysis. All animals administered the drug candidates showed a significant reduction in serum triglycerides and those animals administered either GWA or GWB were observed to have foamy alveolar macrophages and the presence of multilamellar bodies in hepatocytes by electron microscopy. In the plot of the first two principal components, urinary spectra of those animals dosed with GWA or GWB mapped separately to controls, all pre-dose samples and animals dosed with GWC-GWE. Inspection of the principal components loadings indicated an increase in urinary phenylacetylglycine with a concomitant decrease in urinary citrate and 2-oxoglutarate, possibly constituting a novel urinary biomarker set for phospholipidosis. This work exemplifies the use of NMR spectroscopy and pattern recognition methods for the detection of novel biomarker combinations for poorly understood toxicity types and the potential in screening novel drugs for toxicity.(1)H NMR spectroscopy of urine and pattern recognition analysis have been used to study the metabolic perturbations caused following dosing of five novel drug candidates, two of which (GWA, GWB) caused mild lung and liver phospholipidosis, whilst the rest (GWC-GWE) did not cause any detectable toxicity. Urine samples were collected predose, 0-8 h, 8-16 h, 16-24 h and 24-32 h after single, oral dosing with each compound to Han Wistar rats (n = 3 per group), and liver and lung samples for were taken at 48 h for histology. (1)H NMR spectra of whole urine were acquired, processed and subsequently analysed using principal component analysis. All animals administered the drug candidates showed a significant reduction in serum triglycerides and those animals administered either GWA or GWB were observed to have foamy alveolar macrophages and the presence of multilamellar bodies in hepatocytes by electron microscopy. In the plot of the first two principal components, urinary spectra of those animals dosed with GWA or GWB mapped separately to controls, all pre-dose samples and animals dosed with GWC-GWE. Inspection of the principal components loadings indicated an increase in urinary phenylacetylglycine with a concomitant decrease in urinary citrate and 2-oxoglutarate, possibly constituting a novel urinary biomarker set for phospholipidosis. This work exemplifies the use of NMR spectroscopy and pattern recognition methods for the detection of novel biomarker combinations for poorly understood toxicity types and the potential in screening novel drugs for toxicity.

Phenylacetylglycine, a putative biomarker of phospholipidosis: Its origins and relevance to phospholipid accumulation using amiodarone treated rats as a model

Amiodarone was given to male Sprague–Dawley rats at a dose of 150 mg kg−1 day−1 for 7 consecutive days to induce phospholipidosis in the lungs of treated rats. Amiodarone was given alone or concurrently with phenobarbitone. Animals given amiodarone had raised total phospholipid in serum, lung and lymphocytes, and elevated lyso(bis)phosphatidic acid (LBPA) in all tissues. Urinary and plasma phenylacetylglycine (PAG) and hepatic portal:aortal phenylacetate (PA) ratio were increased, whereas hepatic phenylalanine hydroxylase (PAH) activity and plasma phenylalanine:tyrosine ratio were not affected. Phenobarbitone treatment increased hepatic total P450 content and induced 7-pentoxyres-orufin O-dealkylatian (PROD) activity, as expected, but had no effect on any other biochemical parameter. Plasma amiodarone concentration was reduced in rats co-administered both drugs and phospholipid accumulation in target tissues was attenuated compared with rats treated with amiodarone alone. However, phenobarbitone co-administration failed to alter the magnitude of response with regards to urinary PAG excretion and plasma concentration of its precursors after amiodarone treatment. Increased intestinal absorption of PAG precursors probably resulted in the raised urinary PAG after amiodarone treatment. Urinary PAG correlated weakly with serum, lymphocyte and lung phospholipids. However, urinary PAG excretion was similar in rats dosed solely with amiodarone or in combination with phenobarbitone, despite the fact that the degree of phospholipid accumulation was far less in rats given the combined treatment. Nevertheless, urinary PAG was raised only in animals exhibiting abnormal phospholipid accumulation in target tissues and may thus be useful as a surrogate biomarker for phospholipidosis.

Characterisation of a potential biomarker of phospholipidosis from amiodarone-treated rats

A novel and relatively simple analytical method for the separation, characterisation and semi-quantitation of phospholipids (PLs) from extracts of complex biological samples has been developed. This methodology allows PL extracts from cells and tissues to be analysed by liquid chromatography (LC) coupled to electrospray ionisation mass spectrometry (ESI-MS). Complex mixtures of PLs were separated on a high-performance liquid chromatography (HPLC) system using 0.5% ammonium hydroxide in methanol/water/hexane/formate mixture with UV detection at 205 nm. Identification and structural characterisation of molecular species were carried out utilising ESI-MS and MS/MS in the negative ion mode. The abnormal accumulation of PLs (phospholipidosis) was induced in male Sprague-Dawley rats by administration of the cationic amphiphilic drug (CAD), amiodarone. Analysis of the PL profile of liver and lung tissues, lymphocytes and serum from treated rats was carried out using this analytical procedure (LC-ESI/MS/MS). Differences in PL profiles between treated and untreated animals were highlighted by principal component analysis (PCA). This led to the selection of a potential metabolic marker of phospholipidosis (PLD) identified as a lyso-bis-phosphatidic acid (LBPA) derivative, also known as bis(monoglycero)phosphate (BMP). This PL was absent in control animals but was present in quantifiable amounts in all samples from amiodarone-treated rats.

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.

Determination of taurine in biological samples and isolated hepatocytes by high-performance liquid chromatography with fluorimetric detection

A high-performance liquid chromatographic method with fluorimetric detection is described for the routine and selective determination of taurine in urine, serum, tissues and isolated hepatocytes. The preparation and use of ion-exchange resins to extract taurine from biological samples is included. Taurine was derivatised with o-phthalaldehyde/2-mercaptoethanol prior to injection onto a C18 column (LiChrospherR 100 RP-18, 5 microns, 125 x 4 mm I.D.). Isocratic elution of the adduct was carried out using NaH2PO4 (0.05 M, pH 5.4) in methanol and water (43:57, v/v). Homoserine was used as an internal standard to facilitate the standardisation and quantitation of samples and analysis was completed in 6 min with homoserine and taurine eluting after 3 and 4 min, respectively. The method will detect 0.5 pmol of taurine on the column. Appropriate dilutions of these biological samples enable these samples to be assayed on an autosampler, using the same standard curve. Concentrations of taurine in human, dog and rat urine, rat liver, serum and isolated hepatocytes are reported.