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 and alpha-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.

The correlation between urinary and liver taurine levels and between pre-dose urinary taurine and liver damage.

Analysis of data from several studies has shown that urinary taurine levels are highly significantly correlated with liver taurine concentration in control rats. Furthermore, urinary taurine levels measured before dosing with various hepatotoxic agents are significantly correlated with serum AST and ALT values measured after dosing with hepatotoxicants. That is, animals with low urinary taurine values and therefore low liver taurine concentrations tend to show greater hepatic damage. These data suggest that taurine may have a protective function in the liver.

Reduction of liver taurine in rats by beta-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.

Effect of various non-hepatotoxic compounds on urinary and liver taurine levels in rats.

Administration of compounds which alter protein synthesis or sulphur amino acid metabolism in rats results in changes in the excretion of urinary taurine. Treatment with diethylmaleate (DEM) or phorone, which will deplete glutathione (GSH), reduces taurine excretion, whereas treatment with buthionine sulphoximine (BSO), which will inhibit glutathione synthesis, increases taurine excretion. Treatment with cycloheximide, an inhibitor of protein synthesis, increases taurine excretion, whereas pretreatment with phenobarbital, which will increase protein synthesis, decreases taurine excretion. Administration of agents which damage organs other than the liver such as the kidney, heart and testes, does not increase urinary taurine.

A review of documentation requirements for preclinical sections, for marketing submissions in the European Community, Japan and the USA.

Standardization of the documentation requirements for preclinical sections, for marketing submissions in the European Community (EC), Japan and the USA is proposed. It is unnecessary to standardize the leading summaries of submissions as their format is predicated by the way in which the regulatory authorities review applications. Harmonization of the technical sections will reduce the duplication of effort expended by pharmaceutical companies to produce documentation specific for each regulatory authority. Elimination of requirements specific to individual authorities will further reduce the resource requirements. The proposals made in this paper are aimed to increase the 'user-friendliness' of the preclinical documentation and therefore expedite the review process.

Implications of recommendations from the International Conference on Harmonization (ICH) for the safety evaluation of new medicines involving animal studies for the pharmaceutical industry.

Based upon the recommendations given by the ICH outlined in this document, it is proposed that: 1. In rodents, single dose toxicity studies should be conducted using as few animals as possible. 2. Single dose toxicity studies in non-rodents will not be performed except possibly as a component of dose escalation studies. 3. Recovery animals on repeat dose studies will not be monitored for delayed toxicity. 4. Twelve month rodent studies will not be performed. 5. Twelve month non-rodent studies will be performed for chronic therapies except in the few circumstances when the ICH exclusion criteria can be met. 6. EEC protocols for reproductive toxicology studies will be followed until a new guideline is issued. 7. Consultation with regulatory authorities, especially the FDA, may be required to set the doses to be used in carcinogenicity studies.

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.

Animal toxicology studies on new medicines and their relationship to clinical exposure: a review of international recommendations.

Written guidance on the toxicology studies which should be completed to support an application to market a new medicinal product in the EEC, Japan, and the USA is readily available, although the recommendations can vary significantly between the three markets. Less clear are the regulatory authorities' expectations concerning the timing of these studies relative to the clinical development programme. All available guidelines for premarketing toxicology requirements, and the portions thereof needed to support various stages of the clinical trials programme will be reviewed. Significant differences among the three markets and between written guidance and customary practice will be highlighted. The practical implications for the clinical development programme will be identified.

Ondansetron: pre-clinical safety evaluation.

A programme of pre-clinical safety evaluation of ondansetron has been undertaken which involved a series of studies - single dose studies, repeat dose studies, reproduction studies, genotoxicity studies, oncogenicity studies, local irritancy studies, and a hypersensitivity study. Ondansetron was found to have a very good safety profile, and the only toxicity identified was associated with central nervous system activity when near lethal doses were administered. It was not genotoxic and had no reproductive or oncogenic potential.

Studies on hydrazine hepatotoxicity. 1. Pathological findings.

The pathogenesis and factors affecting hydrazine-induced fatty liver have been investigated in rats using histological and ultrastructural examination. A dose of 20 mg hydrazine/kg caused the accumulation of lipid, swelling of mitochondria, and the appearance of microbodies in both periportal and midzonal hepatocytes and in the proximal tubular cells of the kidney. These changes were detectable by light or electron microscopy 24 h after dosing with hydrazine. A dose of 60 mg/kg was the highest dose tolerated for 24 h, but the severity of the fatty liver was similar to that after a dose of 40 mg/kg. The accumulation of lipid droplets and the swelling of mitochondria were detectable by electron microscopy 30 min after dosing, but the accumulation of fat could not be detected by light microscopy until 4 h after dosing. Pretreatment of animals with phenobarbital or piperonyl butoxide respectively reduced and increased the severity of the fatty liver. Pyruvate azine was much less toxic than the parent hydrazine on a molar basis, although some fatty vacuolation was detectable in midzonal hepatocytes.

Studies on hydrazine hepatotoxicity. 2. Biochemical findings.

Hydrazine causes a dose-related increase in liver triglycerides and in liver weight and causes a decrease in hepatic glutathione. The threshold dose for the toxic effect is around 10 mg/kg, and the optimal effect is seen after a dose of 40 mg/kg. The effect of hydrazine on liver weight and glutathione was detectable within 30 min of dosing, but the elevation of hepatic triglycerides was not detectable until 4 h after dosing. At 24 h after a dose of 60 mg hydrazine/kg, hepatic reduced glutathione was approximately 50% of the control value and triglycerides were about 7 times the normal level. In vitro studies indicated that hydrazine is metabolized by rat liver microsomal enzymes, this being dependent on NADPH and oxygen. Pretreatment of animals with phenobarbital or piperonyl butoxide respectively decreases and increases the hepatotoxicity. Prior depletion of hepatic glutathione by administration of diethyl maleate had no effect on the toxicity. Pyruvate azine, a probable metabolite of hydrazine, is much less toxic than hydrazine itself on a molar basis. These and other results suggest that although hydrazine is metabolized via several routes, the hepatotoxicity may well be due to the parent compound rather than a metabolite.

The ultrastructure of the non-neurosecretory components in the brain of the midge, Chironomus riparious Mg. (diptera: nematocera).

The ultrastruct of the neural sheath, glial cells and neurons in the brain of the neoimaginal male Chironomus riparius is described. The neural sheath comprises a neural lamella and underlying perineurium. The neural lamella consists of an amorphous matrix in which fine fibrils occur. The perineurium is composed of two cell types forming a continuous layer around the brain. The subjacent cortical layer, composed of the cell bodies of neurons and glial cells, varies considerably in thickness and surrounds the centrally located neuropiles. Three types of glial cells are distinguished on the basis of their positions and appearances. Five types of neurons are described which differ in size and relative frequency of organelles. Four types of axons, including those of neurosecretory cells, are distinguished by their size and content.