The metabolism of hydralazine in a group of slow acetylator patients [6] with the drug-induced lupus syndrome was compared with the metabolism in asymptomatic control subjects [17]. There were no toxicologically significant difference in metabolite excretion between the groups which reached statistical significance, although there were interesting trends. However, the single lupus patient with the rapid acetylator phenotype excreted considerably greater quantities of phthalazinone than control patients and also increased amounts of hydrazine and hydralazine hydrazones. These results and the trends overall are consistent with the hypothesis that the metabolism of hydralazine may indeed be responsible for the drug induced lupus syndrome

Alcohol was administered chronically to female Sprague-Dawley rats in a nutritionally adequate totally liquid diet for 28 days. This resulted in significant hepatic steatosis and lipid peroxidation. beta -Alanine, when co-administered with alcohol, seemed to increase hepatic steatosis, as assessed histologically, but decreased triglyceride levels as measured biochemically. In addition, beta -alanine and especially alcohol co-administered with p-alanine, significantly increased homocysteine and cysteine excretion into urine throughout the 28-day period of ethanol administration. Serum homocysteine levels were significantly higher in alcohol- and alcohol plus beta -alanine-treated animals compared to pair-fed control animals. Alcohol did not affect the urinary excretion of taurine, except after 21 days, when levels were reduced. Levels of liver taurine were markedly depleted in animals receiving alcohol and particularly alcohol plus beta -alanine, compared to pair-fed controls. Liver and serum taurine levels were also markedly depleted in animals receiving beta -alanine and alcohol plus beta -alanine, compared to non-beta -alanine-treated animals. There was evidence of slight cholestasis in animals treated with alcohol and more so with alcohol plus beta -alanine, as indicated by raised serum alkaline phosphatase and bile acids. These in vivo findings demonstrate for the first time that animals treated with beta -alanine may be more susceptible to ethanol-induced hepatic dysfunction, possibly as a result of taurine depletion.

Stress protein induction has been advocated as a sensitive indicator of compound-induced toxicity. In monolayer cultures of primary hepatocytes, however, the two stress proteins, Hsp25 and Hsp72/3 are up-regulated, probably due to the effect of the isolation procedure and adaptation of the cells to the culture conditions. The aim of the current studies was to determine whether liver spheroids would provide an improved experimental model for the study of heat shock protein induction in vitro. Primary rat hepatocytes were cultured as liver spheroids and the expression of Hsp25 and Hsp72/3 measured along with the levels of ATP, GSH and albumin secretion. Hsp72/3 was initially increased in spheroid culture but returned to in vivo levels after 3 days of culture. Hsp25 was maintained at in vivo levels until day 6 of culture, after which levels increased slightly. The effects of the two hepatotoxins, hydrazine and cadmium chloride (CdCl(2)), were therefore measured on day 6 of spheroid culture. CdCl(2) had no effect on Hsp25 but increased Hsp72/3 at concentrations that affected other biochemical parameters. Hydrazine caused a rapid reduction in ATP levels and albumin secretion, but did not affect Hsp72/3. Hsp25 was slightly induced by hydrazine at later sampling times at concentrations, however, that affected other biochemical parameters. It can be concluded that liver spheroids provide a model for studying stress protein expression. However, the increase in stress proteins appears to be a relatively insensitive parameter compared to other more conventionally used toxicity endpoints and the response appears to vary with individual toxins under study.

Ifosfamide (55 mg kg-1 and 110 mg kg-1) was administered via single i.p. injections to Sprague-Dawley rats and urine samples were collected for the periods of -24-0, 0-8 h, 8-24 h, 24-48 h and 48-72 h post-dose. Quantitative changes in the excretion pattern of small organic molecules in the urine of rats treated with ifosfamide were studied using high frequency 1H NMR spectroscopy. The kidneys and livers of the animals were also examined, but showed no marked histopathological changes. 1

Alcohol (ethanol) was administered chronically to female Sprague-Dawley rats in a nutritionally adequate, totally liquid diet for 28 days. This resulted in significant hepatic steatosis and lipid peroxidation. When taurine was administered for 2 days following alcohol withdrawal it was found to reduce alcohol-induced lipid peroxidation and completely reversed hepatic steatosis. The reversal of hepatic steatosis was demonstrated both biochemically and histologically. Two days following alcohol withdrawal, the apparent activity of the alcohol-inducible form of cytochrome P450 (CYP2E1) was unchanged although total cytochrome P450 content was increased. In addition, alcohol significantly inhibited hepatic methionine synthase activity and increased homocysteine excretion in urine. Although alcohol did not affect the urinary excretion of taurine (a non-invasive marker of liver damage), levels of serum and hepatic taurine were markedly raised in animals given taurine following their treatment with alcohol, compared to animals given taurine alone. There was evidence of slight bile duct injury in animals treated with alcohol and with alcohol followed by taurine, as indicated by raised serum alkaline phosphatase (ALP) and cholesterol. Aspartate aminotransferase (AST) was also slightly raised. The effects of taurine on reversing hepatic steatosis may be due to the enhanced secretion of hepatic triglycerides. It is suggested that increased bile flow as a result of taurine treatment may have contributed to the removal of lipid peroxides. These in-vivo findings demonstrate for the first time that hepatic steatosis and lipid peroxidation, occurring as a result of chronic alcohol consumption, can be reversed by administration of taurine to rats for 2 days.

Changes in urinary levels of taurine have been reported in rats following treatment with various xenobiotics including those which alter protein synthesis and/or are hepatotoxic. This paper reports on the time course of the urinary elevation of taurine following treatment of rats with tetracycline (50, 150 and 200mg.kg-1). Maximum taurine excretion occurred 8–12h following dosing. Serum albumin and total protein were significantly lower after 24h (200mg.kg-1). The increase in urinary taurine was dose-related and reflected in the raised serum levels of taurine 24h after dosing. Serum and urinary protein and [3H]-leucine incorporation into acid precipitable protein in liver and muscle were reduced by tetracycline (100, 150 and 200mg.kg-1) 10h after dosing. The reduction in protein synthesis was correlated with increased urinary and serum levels of taurine at 10h. The use of taurine as a non-invasive marker of protein synthesis is discussed.

The Sertoli cells have been identified as the primary locus for creatine synthesis within the seminiferous epithelium. The purpose of the studies reported here was to examine the effect of modulators of Sertoli cell function on creatine secretion by primary cultures of these cells. Sertoli cell-enriched cultures, maintained in a defined medium, secreted creatine into the incubation medium in a manner that was linear with time over at least 6 h, but which had reached a plateau within 24 h. Secretion was stimulated by physiological and toxicological modulators of Sertoli cell function. Incubation of Sertoli cell-enriched cultures in the presence of FSH (> or = 40 mU ml-1), dibutyryl cyclic AMP (> or = 0.1 mmol l-1), mono-(2-ethylhexyl) phthalate (> or = 1 mumol l-1) or cadmium (> or = 3 mumol l-1) increased the secretion of creatine into the incubation medium by at least 85% over 24 h. Creatine secretion by Sertoli cell-enriched cultures, incubated over 4 h in a balanced salt solution, was independent of exogenous L-glutamine. However, the stimulation of secretion induced by 1 mmol dibutyryl cyclic AMP l-1 was dependent on the presence of 4 mmol L-glutamine l-1 in the incubation medium, which suggests that an increase in creatine secretion occurs as a consequence of stimulated glutamine oxidation.

Alcohol was administered chronically to female Sprague Dawley rats in a nutritionally adequate totally liquid diet for 28 days. This resulted in hepatic steatosis and lipid peroxidation. Taurine, when co-administered with alcohol, reduced the hepatic steatosis and completely prevented lipid peroxidation. The protective properties of taurine in preventing fatty liver were also demonstrated histologically. Although alcohol was found not to affect the urinary excretion of taurine (a non-invasive marker of liver damage), levels of serum and liver taurine were markedly raised in animals receiving alcohol + taurine compared to animals given taurine alone. The ethanol-inducible form of cytochrome P-450 (CYP2E1) was significantly induced by alcohol; the activity was significantly lower than controls and barely detectable in animals fed the liquid alcohol diet containing taurine. In addition, alcohol significantly increased homocysteine excretion into urine throughout the 28 day period of ethanol administration; however, taurine did not prevent this increase. There was evidence of slight cholestasis in animals treated with alcohol and alcohol + taurine, as indicated by raised serum bile acids and alkaline phosphatase (ALP). The protective effects of taurine were attributed to the potential of bile acids, especially taurine conjugated bile acids (taurocholic acid) to inhibit the activity of some microsomal enzymes (CYP2E1). These in vivo findings demonstrate for the first time that hepatic steatosis and lipid peroxidation, occurring as a result of chronic alcohol consumption, can be ameliorated by administration of taurine to rats.

The testis synthesizes creatine from both arginine and glycine precursors, but when rat testicular tissue is separated into seminiferous tubules and interstitial cells, creatine synthesis occurs only in the tubular fraction. The purpose of the work presented here was to define the locus of creatine synthesis within the seminiferous tubules, by using cell separation and culture techniques to examine synthesis in the Sertoli cells and germ cells. The total creatine content, in the cellular compartment and incubation medium, of Sertoli-germ cell co-cultures and of Sertoli cell-enriched cultures, largely free of germ cells, increased by similar amounts over a 24 h incubation period. Sertoli cell-enriched cultures incorporated radioactivity from L-[guanidino-14C]arginine and [1-14C]glycine into both creatine and its biosynthetic precursor, guanidinoacetic acid. Isolated germ cells did not incorporate radioactivity from L-[guanidino-14C]arginine into either creatine or guanidinoacetic acid when incubated at a similar density and protein concentration under similar conditions. It is concluded that the synthesis of creatine observed in isolated rat seminiferous tubules occurs within the Sertoli cells and not the germ cells.

Methylene dianiline (DAPM) causes hepatic damage and bile duct necrosis in rats. This has been detected histologically and biochemically. The toxicity was dose related over the range 0-100 mg/kg but the dose response relationship showed a maximum at about 75-100 mg/kg. This was true for both histopathology and biochemical parameters of liver dysfunction. When animals were depleted of taurine using beta-alanine pretreatment, the toxicity of DAPM was increased. Conversely treatment of rats with taurine, significantly attenuated the rise in alanine transaminase (ALT). However depletion of taurine with guanidinoethanesulphonate (GES) attenuated rises in both transaminases. It is concluded that taurine may play a role in the toxicity of DAPM but that GES, although depleting taurine as does beta-alanine, causes additional effects such as increasing glutathione (GSH), perhaps leading to protection.

Studies have been carried out in rats in vivo and in isolated hepatocytes from the same strain of rat in vitro using the hepatotoxicant hydrazine as a model compound. These studies have shown that a number of biochemical changes occur and are measurable in both systems. However, despite measuring the same parameters in each system, the effects do not necessarily show a quantitative or qualitative correlation. Thus depletion of glutathione and ATP occurred in both systems but required a much higher concentration in vitro. The effects on more liver-specific parameters such as triglyceride, citrulline and taurine levels in vivo were different or not observed in vitro and the inhibition of urea synthesis and cytotoxicity in vitro were not observed in vivo, although these endpoints are more relevant markers of hepatic effects. Inhibition of protein synthesis proved to be the marker that showed the best correlation, occurring at a similar concentration in vitro as in vivo, although not to the same extent. The importance of identifying specific endpoints of toxicity, the problems of comparing in vivo with in vitro data and the limitations in their interpretation are highlighted by the data presented. A knowledge of the underlying mechanisms of toxicity will clearly facilitate the design and interpretation of specific in vitro biomarkers.

Repeated oral administration of salbutamol to lambs for 28 days was found to decrease levels of taurine significantly in the serum and heart, and the mean excretion of taurine into urine was significantly less than in controls, Serum urea, low density lipoprotein and high density lipoprotein were also significantly reduced. Consistent with these changes, fat content in muscle was reduced, whereas protein content was not significantly changed, Body weight was not significantly changed by salbutamol treatment but heart and kidney weights (relative to body weight) were significantly increased, Salbutamol excretion in urine was relatively constant and residues were detected in certain organs and tissues, notably liver, bile and kidney, Changes in urinary and serum taurine level may reflect subtle changes in protein metabolism not detectable as changes in body weight or gross protein content.

The synthesis of taurine from N-acetylcysteine has been examined in rats in vivo and in rat hepatocyte suspensions in vitro. In rats in vivo, administration of N-acetylcysteine significantly increased urinary taurine (3 fold) 24h after dosing and liver glutathione levels. Liver taurine was not increased significantly. In hepatocytes incubated in the presence of N-acetylcysteine, glutathione concentration increased to a maximum after 1 hour but the increase was not dependent on the concentration of N-acetylcysteine. In contrast, after an initial lag phase, taurine synthesis increased in relation to the concentration of N-acetylcysteine and continued for 3 hours. Glutathione synthesis seems to be preferential to taurine synthesis. Taurine synthesis from cysteine sulphinate was greater and from hypotaurine was greatest and maximal after 1 hour. Implications for the mechanism of protection by N-acetylcysteine are discussed.

We have shown that urinary taurine level may be used as a biomarker of pathological and biochemical lesions. Detection of changes in the urinary concentration of this low molecular weight metabolite indicates biochemical lesions which may also be associated with pathological damage. Hepatotoxic compounds such as CCl4, galactosamine and thioacetamide that cause hepatic necrosis and compounds such as hydrazine and ethionine that cause fatty liver all result in elevated urinary taurine levels in rats. However compounds which do not cause liver damage, such as cycloheximide, also raise urinary taurine levels. All of these substances are known to or are believed to inhibit protein synthesis. Conversely, compounds which increase protein synthesis, such as phenobarbital and clenbuterol, significantly decrease urinary taurine levels. Compounds which interfere with hepatic GSH synthesis will also change urinary taurine levels. Thus, depletion of GSH with diethyl maleate or phorone decreases urinary taurine whereas inhibition of GSH synthesis with compounds such as buthionine sulphoximine increases urinary taurine levels. In isolated hepatocytes in vitro, leakage of taurine occurs in response to cytotoxic compounds such as hydrazine and allyl alcohol. However, total taurine levels were increased by the hepatotoxicant CCl4. Taurine synthesis is decreased by depletion of GSH with allyl alcohol in isolated hepatocytes. Therefore taurine levels are an important potential biomarker for biochemical lesions induced by chemicals both in vivo and in vitro, in particular changes in protein and GSH synthesis.

Administration of clenbuterol to rats in the drinking water over a 4 day period increased incorporation of [3H]leucine into muscle protein and caused a slight reduction in urinary 3-methylhistidine but did not result in an increase in body or muscle weight. However, both urinary and liver taurine were significantly reduced at the highest dose of clenbuterol (2 mg.kg-1.day-1). Serum creatine kinase, muscle isoenzyme (CK-MM) was raised and single muscle fibre injury was observed in the soleus muscle in animals treated with the middle dose (0.2 mg.kg-1.day-1) and highest dose (2 mg.kg-1.day-1). The reduction in the body pool of taurine caused by clenbuterol is of concern as taurine has been shown to have protective properties.

We have shown that urinary taurine and creatine may be used as biomarkers of pathological and biochemical lesions. Detection of changes in the urinary concentration of these two low-molecular-weight metabolites indicates biochemical lesions which may also be associated with pathological damage. Thus changes in protein or glutathione metabolism will lead to changes in urinary taurine. Hepatotoxicants which cause necrosis, but do not affect glutathione status or reduce bile flow, cause taurinuria and creatinuria. Hepatotoxicants which cause fatty liver only cause taurinuria. Testicular toxicants only cause creatinuria. Compounds which result in increased glutathione synthesis, stimulate protein synthesis or reduce bile flow decrease urinary taurine. A combination of the two markers, therefore, can be used to help diagnose a variety of biochemical and pathological lesions.

1. Hepatocytes, isolated from the control, diethyldithiocarbamate (DEDC), acetone, isoniazed and hydrazine pretreated rat, were incubated with hydrazine (8-20 mM) for 3 h. Hydrazine caused a dose-dependent loss of viability, leakage of LDH, depletion of GSH and ATP and an inhibition of the incorporation of 3H-leucine into protein. 2. Pretreatment with DEDC increased, whereas hydrazine and acetone pretreatments decreased the cytoxicity and biochemical effects of hydrazine. Pretreatment with isoniazid slightly increased hydrazine cytotoxicity. Acetone pretreatment reduced the inhibition of protein synthesis caused by hydrazine compared to the control. 3. 4-Nitrophenol hydroxylase activity (P4502E1) correlated with viability, LDH leakage, ATP and GSH depletion in cells from the control, DEDC, acetone and hydrazine pretreated rats. 4. The activities of PROD (P4502B1) and EROD (P4501A1/1A2) also correlated with the above parameters for all treatments. The results suggest that three isoenzymes may be involved in the detoxication of hydrazine. Protein synthesis inhibition did not correlate with the activities of any of the enzymes measured.