The effect of taurine depletion by beta-alanine treatment on the susceptibility to ethanol-induced hepatic dysfunction in rats.

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 beta-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.

The use of liver spheroids as an in vitro model for studying induction of the stress response as a marker of chemical toxicity.

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.

Urinary creatine as a biochemical marker of chemical induced testicular damage.

Urinary creatine has been shown to markedly increase in rats following testicular damage caused by toxicants as diverse as cadmium, 2-methoxyethanol, 1,3-dinitrobenzene, and 2,3,5,6-tetramethylphenylene diamine. More recent findings have shown that urinary creatine is raised in mice exposed to 2-methoxyacetic acid. The most recent studies have revealed that urinary creatine and creatine in interstitial fluid in the testis are raised as early as four hours after dosing with 2-methoxyethanol. Using the testicular toxicants 2-methoxyethanol and cadmium, the authors compared urinary creatine with other markers of testicular damage, such as histopathological assessment of testis by light microscopy, testis weight and lactate dehydrogenase C4 isoenzyme, and testosterone. Urinary creatine was found to be the most sensitive indicator of testicular damage detected by histopathology after both 2-methoxyethanol and cadmium exposure. It is therefore a potentially very useful non-invasive biomarker of male reproductive dysfunction caused by chemicals.

Reversal of ethanol-induced hepatic steatosis and lipid peroxidation by taurine: a study in rats.

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.

Effect of hydrazine upon vitamin B12-dependent methionine synthase activity and the sulphur amino acid pathway in isolated rat hepatocytes.

The effect of the industrial chemical, hydrazine (4-12 mM), on methionine synthase (EC 2.1.1.13) activity and levels of the sulphur amino acids homocysteine, cysteine, and taurine as well as GSH were investigated in vitro in isolated rat hepatocyte suspensions and monolayers in order to explain some of the adverse in vivo effects of hydrazine. None of the concentrations of hydrazine were overtly cytotoxic in hepatocyte suspensions (measured as lactate dehydrogenase [LDH] leakage) after 3 hr. However, after 24 hr in culture cells treated with 12 mM, hydrazine showed a significant increase in LDH leakage. Methionine synthase activity was reduced by hydrazine (8 and 12 mM) in suspensions (by 45 and 55%, after 3 hr) and monolayers (12 mM; 65-80% after 24 hr). This was not due to nitric oxide production and the inhibitor of nitric oxide synthase, Nomega-nitro-L-arginine, failed to protect against the hydrazine-induced loss of ATP and GSH and the reduction in urea synthesis at 24 hr. Homocysteine export was increased by 6 mM hydrazine, and total taurine content of treated cells was increased by 12 mM hydrazine. Thus, hydrazine was found to have several important and possibly deleterious effects on some parts of the sulphur amino acid pathway.

Ethionine toxicity in vitro: the correlation of data from rat hepatocyte suspensions and monolayers with in vivo observations.

The hepato-steatogenic compound ethionine has been used to investigate the correlations between in vivo and in vitro toxicity data. The aim was to find a suitable model of toxicity in hepatocyte suspensions or monolayers in vitro, which could predict the known toxicity of ethionine in vivo and which could be implemented in screening compounds of unknown toxicity. Thus a variety of markers of cytotoxicity, metabolic competence and liver-specific functions were investigated in rat hepatocyte suspensions and monolayers and compared with in vivo data in the rat. The following markers were measured in the appropriate system: (1) Neutral red uptake; 3-(4,5 dimethyl)thiazol-2-yl,-2,5-diphenyl tetrazolium bromide (MTT) reduction; lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) leakage (cytotoxicity). (2) ATP levels, protein synthesis and glutathione (GSH) levels (metabolic competence). (3) Urea and triglyceride synthesis and beta-oxidation (liver specific functions). Ethionine (0-30 mM) did not affect the markers of direct cytotoxicity, except neutral red uptake, which was reduced by 18 and 30 mM ethionine after 20 h in culture. ATP and GSH depletion occurred in hepatocyte suspensions at the highest concentrations of ethionine (20 and 30 mM) after 1 h. In monolayers, GSH levels were reduced after 4 h, but not 20 h. Urea synthesis was increased in hepatocyte suspensions from 1 to 3 h by 10-30 mM ethionine and reduced after 20 h in cultured hepatocytes (18-30 mM). Protein synthesis was reduced and beta-oxidation was increased in ethionine-treated hepatocyte suspensions. Unfortunately, there was no measurable effect on triglyceride accumulation within cells (the major biochemical change in vivo) in either system. Ethionine treated hepatocytes in suspension showed the same rate of triglyceride synthesis and transportation out of cells as control cells. Thus, hepatocyte suspensions were able to mimic the early biochemical effects of ethionine in vivo (ATP and GSH depletion, inhibition of protein synthesis) and some effects on urea synthesis, but monolayer cultures appeared to be less sensitive to the toxicity of ethionine. However, neither in vitro system was able to model the effects of ethionine on the accumulation of triglycerides in vivo.

Biomarkers in toxicology.

The use of biomarkers in toxicology is becoming increasingly important. This article briefly reviews some of the aspects in an attempt to give an overall view of the field. Some of the new developments, particularly in relation to biomarkers of exposure and response, are mentioned. Specific DNA and protein adducts can now be used as biomarkers of the effective exposure so incorporating variations in environmental levels and individual disposition. Analysis of urinary metabolite profiles by NMR can highlight novel markers and allow recognition of patterns of metabolite changes as biomarkers of a toxic response. Novel urinary markers for liver and testicular dysfunction are discussed. Finally, the acetylator phenotype as a biomarker of susceptibility is described.

Creatine metabolism in the seminiferous epithelium of rats. I. Creatine synthesis by isolated and cultured cells.

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.

Creatine metabolism in the seminiferous epithelium of rats. II. Effect of modulators of cellular biochemical function on creatine secretion by cultured Sertoli cells.

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.

The effects of the beta 2-agonist drug clenbuterol on taurine levels in heart and other tissues in the rat.

The administration of a single subcutaneous dose of clenbuterol to rats altered the level of taurine in certain tissues. Taurine levels in cardiac tissue were significantly decreased 3 h after the administration of 250 micrograms/kg of clenbuterol and remained significantly depressed at 12 h post-dose only returning to control values by 24 h. The level of taurine in the liver increased 3 h after clenbuterol administration but was lower than the control value at 24 h post dose. Lung taurine levels were significantly lower than the control value at 12 hr post dose and remained depressed until 24 h post dose. Clenbuterol caused a significant increase in taurine levels in serum and muscle at 3 and 6 hr postdosing respectively but not at other time points. Serum creatine kinase (CK), activity was slightly but significantly raised at the 12 and 24 h time point. The effects of clenbuterol on tissue taurine content were not dose-dependent over the range studied (63-500 micrograms/kg). However taurine levels in the lung were significantly reduced at all doses and in the heart were significantly lower in the treated groups at all except the lowest dose, 12 h post dosing. Liver taurine levels were significantly increased at the highest dose of 500 micrograms/kg. The reduction of taurine concentrations in the heart, caused by clenbuterol, is of concern as taurine has been shown to have protective properties in many tissues especially the heart.

Taurine: protective properties against ethanol-induced hepatic steatosis and lipid peroxidation during chronic ethanol consumption in rats.

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.

Is there a correlation between taurine levels and xenobiotic-induced perturbations in protein synthesis?: a study with tetracycline in rats.

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 200 mg.kg-1). Maximum taurine excretion occurred 8-12 h following dosing. Serum albumin and total protein were significantly lower after 24 h (200 mg.kg-1). The increase in urinary taurine was dose-related and reflected in the raised serum levels of taurine 24 h 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 200 mg.kg-1) 10 h after dosing. The reduction in protein synthesis was correlated with increased urinary and serum levels of taurine at 10 h. The use of taurine as a non-invasive marker of protein synthesis is discussed.

Modulation of taurine levels in the rat liver alters methylene dianiline hepatotoxicity.

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.

Effect of fructose on the biochemical toxicity of hydrazine in isolated rat hepatocytes.

Rat hepatocyte suspensions were incubated with various concentrations of hydrazine (0, 8, 12, 16, 20 mM) for 1, 2 and 3 h. In some experiments fructose (10 mM) was added either during the preincubation period, or 1 h after the start of hydrazine treatment. In certain experiments in which fructose was added, the glycolytic inhibitor sodium fluoride (3 mM) was also added during the preincubation period. Hepatocytes incubated with hydrazine alone demonstrated both a concentration- and time-dependent loss of cell viability as measured by increased Trypan blue uptake and lactate dehydrogenase (LDH) leakage. These parameters were reduced and delayed by fructose when added either before or 1 h after hydrazine treatment. There was also both a concentration- and time-dependent loss of ATP and reduced glutathione (GSH) content with hydrazine treatment. Moreover, fructose caused an initial rapid depletion of ATP but thereafter ATP levels were increased in control hepatocytes. Fructose reduced both the depletion of ATP and GSH in hydrazine- treated hepatocytes. Urea synthesis was inhibited by all concentrations of hydrazine studied but fructose treatment after 1 h did not alter this. This study also demonstrated that fluoride, an enolase inhibitor, abolished the protection against depletion of ATP levels provided by fructose, without affecting cell viability or GSH levels. These findings suggest that the cytotoxicity of hydrazine and its effects on urea synthesis and GSH levels are not a direct result of ATP depletion. The protective effects of fructose against the cytotoxicity may be due to a direct interaction with hydrazine.

Changes in taurine levels in response to repeated administration of the beta 2-agonist salbutamol in lambs.

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.

Correlations between in vivo and in vitro effects of toxic compounds: Studies with hydrazine.

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.

Triglyceride disposition in isolated hepatocytes after treatment with hydrazine.

Treatment of animals with hydrazine causes the accumulation of triglycerides in the liver but the mechanism remains unclear. Therefore, the effect of hydrazine on hepatic triglyceride synthesis and subsequent transport was studied in a hepatocyte model, in vitro in order to isolate liver cells from extrahepatic influences. Hepatocytes were isolated and either incubated in suspension with [14C]palmitate in the presence of hydrazine (2-12 mM) or pre-incubated with [14C]palmitate, washed free of the fatty acid and then incubated with hydrazine (2-12 mM). Hydrazine resulted in a significant reduction in the incorporation of [14C]palmitate into triglycerides and reduction in the transportation of triglycerides out of cells. When [14C]palmitate was in the incubation medium, ATP levels were reduced by lower concentrations of hydrazine than have previously been reported. None of the concentrations of hydrazine used affected cell membrane integrity (viability) as measured by LDH leakage. The 14CO2 produced by the beta-oxidation of [14C]palmitate was also measured in short term incubations (30 min) carried out in sealed vessels. There was a dose dependent increase in 14CO2 produced by very low concentrations of hydrazine (0.01-0.1 mM) after which the effect was maximal and concentrations above 8 mM hydrazine decreased 14CO2 production. The data suggest that the inhibition of transportation of triglycerides out of cells by hydrazine may have a more important role in the accumulation of triglycerides in the liver than has been previously recognised. However, the model was not able to mimic the accumulation of triglycerides in hepatocytes seen in vivo.

Urinary creatine as a potential marker of testicular damage: effect of vasectomy.

The concentrations of creatine in the rat testis are high compared to all other tissues except muscle. The total creatine content of the testis is also considerably greater than all organs examined except muscle. Vasectomised male rats were given 2-methoxyethanol or cadmium chloride at doses that caused testicular damage. In the vasectomised rats, testicular damage still resulted in significant creatinuria. This indicates the creatine reaches the urine at least partly via the blood stream and not via the vas deferens.

Changes in taurine as an indicator of hepatic dysfunction and biochemical perturbations. Studies in vivo and in vitro.

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.