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The effects of age and different diets on the activity of hepatic drug-metabolizing enzymes have been studied in rats. The enzymes studied were biphenyl 2- and 4-hydroxylase, p-nitrobenzoate reductase, 4-methyl-umbelliferone glucuronyltransferase, and cytochrome P-450 which is regarded as the major terminal mixed-function oxidase in hepatic microsomal hydroxylations. The activity of these enzymes was determined at various ages between 6 and 100 days postnatum. The activity of each enzyme rose to a peak at a different rate. The peak was reached soon after weaning, and activity then decreased with age to the mature level. With biphenyl 2-hydroxylase, the activity fell rapidly after 31 days and disappeared altogether at 70 days. During the first three weeks of postnatal life, undernutrition did not result in any significant change in the activity per g. liver weight of the enzymes studied. The total activity of the enzymes per liver was, however, lower, in the livers of the undernourished animals due to the smaller size of the liver. In male weanling rats pair fed with respect to proteins with additional calories fed for 7, 14 and 28 days, the hepatic concentration of the enzymes was, in general increased. The activity of glucuronyl transferase was, however, depressed in the absence of additional calories and cytochrome P-450 was depressed in the presence of additional calories. In the animals fed the protein deficient diet with additional calories, the rise in the activity of biphenyl 4-hydroxylase was sufficient to compensate for the lower liver weight so that the absolute amount per liver was the same as in the controls. The adaptive response of this enzyme to the protein deficient diet in the in vitro studies was further suggested by the finding that the capacity of the body to metabolize biphenyl and excrete it as hydroxy-biphenyl in the urine was similar to that of control animals. Any possibility of a conformational change of the enzyme site being responsible for the adaptive response of the enzyme has been excluded. However, a correlation between the increase of biphenyl 4-hydroxylase activity and plasma corticosteroid level in protein deficient animals has been observed. The possibility of the adaptive response being mediated by corticosteroids has further been supported by the increase in the concentration of biphenyl 4-hydroxylase as a result of corticosterone administration in well-fed animals. The current investigation has been extended by a study of the effect of substitution of starch by sucrose, glucose, fructose or an equimolar mixture of glucose and fructose on the activity of drug-metabolizing enzymes in growing rats. The presence of sucrose in the diet at concentrations of either 60% or 10% depressed activity of biphenyl 2- and 4-hydroxylases and level of cytochrome P-450. Other disaccharides, such as lactose or maltose did not have the same effect.

The recognition and subsequent demonstration, in vitro, of the alkylating potential of dichlorvos has led to a suspicion that this triester of phosphoric acid may be a mutagen and a carcinogen. Despite consistently negative results obtained in mammalian mutagenicity and carcinogenicity tests it was never-the-less important to investigate the alkylating reactivity in vivo. In the experiments described in this thesis, male CFE rats were exposed to atmospheres containing 0,064ug 1[-1] of [Me-[14]C] dichlorvos (113Ci.mol[-1]) for 12 hours. Analysis of the DNA from the total soft tissues from 20 rats revealed no methylation at the N-7 atom of guanine moieties. A comparative study with [methyl-[14]C]methanesulphonate gave rise to a readily detectable extent of methylation of the N-7 atom of the guanine moieties in DNA. The limit of detection of methylation of the DNA in the dichlorvos study was one methyl group per 5.7 x 10[11] nucleotide units. The proportion of the administered dose that would be consumed in this hypothetical reaction would be 0.000001%. The exposure period employed in this study (12h) constituted a significant fraction of the half-live of 7-methylguanine moieties in DNA (3 days) and the current findings therefore indicate that dichlorvos would not methylate the DNA of mammalian tissues even when it is inhaled continuously for protracted periods of The administration of radiolabelled adenine, guanine, methionine and formate to otherwise untreated rats, gave rise to the excretion of radiolabelled methylated purines in urine. This finding indicates that the detection of radio- labelled methylated purines, per se, in the urine of animals exposed to methyl-labelled methylating agents does not constitute evidence for the spontaneous methylation of the purine moieties of nucleosides and nucleic acids by methylating agents, in vivo.

Intact hepatocytes have been prepared from a number of adult mammalian species by a procedure involving digestion of the finely sliced liver in a mixture of collagenase and hyaluronidase enzymes. The cell yield, viability index and ability of the hepatocytes to form non-proliferating monolayer cultures was assessed. This procedure was also applied to the isolation of viable renal tubule fragments from several species. The metabolism of a number of aromatic compounds, namely biphenyl, 7-ethoxycoumarin, benzo(a)pyrene and 4-methylumbelliferone was studied in hepatocytes isolated from the rat, ferret and hamster. Overall, hamster hepatocytes exhibited the greatest degree of mixed function oxidase and UDP-glucuronyltransferase activity. Rat hepatocytes were superior to ferret hepatocytes in metabolising capability. The ferret hepatocytes were generally less efficient at conjugation, particularly with glucuronic acid. Renal metabolic activities were considerably less than those of liver. The metabolism of benzoic acid was studied in liver and renal cell preparations from the rat,hamster, ferret and dog. Benzoic acid was conjugated extensively with glycine by hepatocytes and renal cells of the rat and hamster, whereas, the renal cells alone, performed this reaction in the dog and ferret. Hepatocytes from these animals showed a limited ability to form benzoylglucuronide. The metabolism of [[3]H]-benzo(a)pyrene (BP) was investigated more fully in the hepatocyte suspensions and cultures. In the rat hepatocyte suspensions, significant differences in the distribution of the metabolites between the cells and extracellular medium were observed. Phenols accumulated intracellularly and only small amounts were released into the medium. Sulphate esters of the phenols also accumulated but to a lesser extent. 4,5-Dihydrodiol and 7,8-dihydrodiol were distributed more evenly between the cells and medium whereas 9,10-dihydrodiol was found mainly in the medium. Conjugates of the phenols, 4,5-dihydrodiol, 7,8-dihydrodiol and 9,10-dihydroxyBP (catechol) were detected in the extracellular medium. 7,8,9,10-Tetrahydrotetrol was identified as a further metabolite of 7,8-dihydrodiol. Low amounts of radioactivity were bound to cellular macromolecules. Hamster hepatocytes formed significantly greater amounts of 4,5-dihydrodiol and a quinone, probably 3,6-quinone, than the rat. Ferret hepatocytes metabolised BP in a manner comparable to the rat but the proportion of conjugates formed was lower and the primary products were not retained to such a degree within the cells. The extent of BP metabolism by the cultured hepatocytes was only 10-20% of that measured in the freshly isolated cells although the metabolic profile was the same. After 3-4 days, the hepatocyte cultures become overrun with fibroblasts which could metabolise BP in a manner comparable to the hepatocytes. Pure fibroblast cultures were more susceptible to the toxicity of BP but when grown in the presence of hepatocytes, this toxicity was significantly reduced. Hepatocytes exposed to BP in culture, showed increased levels of NADPH-diaphorase, succinic dehydrogenase and leucylnaphthylamidase activity; the activity of glucose-6-phosphate dehydrogenase was inhibited and DNA repair synthesis was increased.

The effects of the natural antioxidant vitamin E and synthetic antioxidant ethoxyquin on the ageing process were studied in rats from 21 to 702 days after birth. Two dose levels of vitamin E, 0.05% and 0.5%, and ethoxyquin 0.5%, were used in the diet. In the long term study, the body weight gain of animals receiving diets containing both 0.05% and 0.5% vitamin E were greater than those of either pair-fed or ad-libitum fed controls and this suggests that the control diet was suboptimal with respect to its vitamin E content although the symptoms of vitamin E deficiency were not present. Early reduction in body weight gain in the 0.5% ethoxyquin-treated group was due to impaired palatability rather than impaired food utilisation because a similar growth rate was seen in the pair-fed controls. Later, however, the fall in the food intake and body weight correlated with the occurence of kidney damage. The liver and kidney weight of ethoxyquin-treated animals were significantly higher than in pair-fed controls. The kidney of the ethoxyquin-treated animals in the long term study showed chronic nephritis. In the short term study of two months, 0.5% ethoxyquin in the diet did not show nephrotoxicity. No abnormalities were seen in the kidneys of animals receiving vitamin E in the diet. The histology of bones of rats fed ethoxyquin for a long time suggested less deposition of calcium. In the short term study the humerus of ethoxyquin-fed rats contained a significantly lower level of calcium than in pair-fed controls and the bone weight was also significantly lower. In the long term study of antioxidants, both 0.5% vitamin E and ethoxyquin in the diet caused a significant reduction in PCV and a significant increase in the circulating reticulocytes. Ethoxyquin also significantly increased the clotting time. In the short term study, animals receiving the diets containing 0.05% vitamin E and 0.5% ethoxyquin contained significantly more reticulocytes than pair-fed controls. The clotting time was higher in antioxidant-treated animals, but the difference only reached statistical significance in the case of the 0.5% vitamin E treated animals. The accumulation of lipofuscin pigment in the brain of rats in the long term study shoved a significantly lover intensity of fluorescence in animals that received both 0.5% vitamin E and ethoxyquin in the diet, in spite of the fact that the entry of [14]C ethoxyquin into the brain seemed to be small. The possible relevance of this work to longevity and the ageing process is discussed.

The effects of protein-energy malnutrition on the concentrations of free amino acids, serotonin and norepinephrine in the brains of rats have been studied. The concentrations of glutamic acid, aspartic acid, GABA, glutamine and serotonin have been measured in the forebrain, cerebellum and brainstem of the rat between 7 and 120 days of age. DNA content was studied as a measure of cell number. Maternal protein deficiency from the 5th day of gestation did not result in any significant changes in the concentrations of glutamic acid, aspartic acid, GABA, serotonin and norepinephrine in the whole brain of one day old rats. Pre- and post-natal maternal protein deficiency, on the other hand, resulted in an increase in the concentration of aspartic acid and a decrease in that of glutamic acid in the forebrain, and an increase in the concentration of aspartic acid and in that of GABA in the cerebellum of offspring at 21 d of age. The activity of the enzyme glutamic acid decarboxylase was higher in the cerebellum of the malnourished rats at this age. The entry of U-C[14]-D-glucose was depressed in the forebrain and cerebellum in these animals. The free amino acid concentration in the brain returned to normal after rehabilitation for 120 d whereas the DNA content and cholesterol concentration did not. In male weanling rats fed a low protein diet for 56 days, the composition of the free amino acid was changed. There were differences between the different parts of the brain but the concentration of histidine and methionine were increased in each part studied. In rats fed a high protein diet of equal energy content, the free amino acid pool in the three brain parts was also altered, but not in the same way as in the animals that had eaten the low protein diet. The in vivo uptakes of C[14]-methionine and H[3]-histidine were increased. The incorporation of C[14]-methionine into brain protein was also increased. Giving a low protein diet to weanling rats also depressed the serotonin concentration in the forebrain and brainstem. The in vivo uptake of H[3]-tryptophan into the brain and its incorporation into brain protein was depressed in rats on a low protein diet, but not in those on a restricted high protein diet. Insulin injection enhanced the uptake of H[3]-tryptophan and its incorporation into brain protein in low protein animals. The possible relevance of this work to brain function and to the malnourished child is discussed.

In vitro cultures of guinea-pig lymph node cells were stimulated to transform with phytchaemagglutinin (or antigen) and the incorporation of 3H-glucosamine, -leucine , 3H-uridine and 3H-thymidine measured. The inhibitory effect of established anti-inflammatory drugs and novel candidate compounds on some of these parameters and on cell viability was determined. FPL52806 affected the following measured parameters of stimulated cells, in decreasing order of sensitivity: -glucosamine incorporation at 40h ≈ [[3]H]-thymidine incorporation at 40h > 14C-leucine incorporation at 40 h. ≈ reduction in number of transformed cells > reduction in number of viable cells > 3H-uridine incorporation at 20h. > 3H-uridine incorporation at 1h. The effect of FPL52806 on 3H-thymidine and 14C-leucine incorporation were not due to interference with the binding of phytohaemagglutinin to the cells and were related to the length of time the cells had been exposed to the compound. FPL52806-treated cells were studied by scanning electron microscopy. The potencies of 6,8-di-alkyl chromones as inhibitors of 3H-thymidine and [[14]C]-leucine incorporations were shown to be mathematically related to the lipophilicities of the compounds. The extent to which these compounds bound to bovine serum was also related to the lipophilicity of the compounds. The order of decreasing potency of established anti-inflammatory drugs as inhibitors of 3H-thymidine incorporation was chloroquine, prednisolone, flufenamic acid, phenylbutazone, indomethacin, ibuprofen and sodium salicylate. This potency order is discussed with respect to the therapeutic potencies of the drugs in rheumatoid arthritis and to the activities of the drugs in other in vitro and in vivo test systems which may detect anti-inflammatory properties. The advantages of the stimulated lymph node cell system as anti-inflammatory screen are described. The biology of the lymphocyte and the evidence for the involvement of this cell in rheumatoid arthritis is described.

The effect of molecular modification on the kinetics of absorption, distribution, metabolism and excretion of selected carbamates has been interpreted in terras of changes in physicochemical properties. The use of a series of aliphatic homologues of general structure R-O-CO-NH[2] was the basis for this work since these compounds show regular incremental changes in their apparent partition coefficients, aqueous solubilities, and ability to bind with macromolecules. Modification of the R group (eg branching of the aliphatic side chain or introduction of an aromatic group ) and N-methylation have also been studied as independant factors to elucidate the role of additional physico-chemical factors. The intestinal absorption of the homologous series was investigated using both in vitro and in situ techniques. An increase in absorption rate was observed when the chain length was increased from methyl to n-butyl. Further increase in the chain length resulted in a fall in the absorption rate. The absorption process appeared to be passive and no metabolism by the gut wall was detected. High tissue levels of carbamate were found using the in vitro system; especially with the more lipophilic carbamates. This phenomenon was not apparent using the in situ procedure. A similar lipophilicity parabola" was established with a series of N-methylated carbamates. These results have been discussed in terms of a two compartment model where both a hydrophilic barrier and a lipoidal membrane control the rate of intestinal absorption. The gastric absorption of the same two series of carbamates has been studied in situ. A good linear correlation betv/een the absorption rate constant and the partition coefficient over a range of 0.1 to 1000 was found. This has been contrasted with the absorption trends observed with the small intestine and it would appear that the role of the hydrophilic barrier is not as marked in controlling absorption rates from the stomach as it is in the intestine. Further comparison between the two organs have been made with respect to the role of steric factors and hydrogen bonding and to the effect of the non-ionic detergent Tween 80. The intestinal absorption behaviour of the weak acidic drug Carbenoxolone was also found to confirm to the "lipophilicity parabola". A pharmacokinetic profile of two carbamate insecticides---Carbaryl and Landrin---has been carried out. Despite their similar partitioning properties and absorption rates, these two compounds showed very different distribution and metabolism kinetics. Comparison of the half-lives of elimination of carbamate from plasma with half-lives of CO2 production showed that hydrolysis of the carbamate moiety proceeded through at least one intermediate.

1. The published work on the behaviour of trichlorofluoromethane in biological systems is discussed and compared with carbon tetrachloride and halothane. 2. The possible metabolism of trichlorofluoromethane by liver microsomal preparations in vitro under aerobic and anaerobic conditions has been investigated. Evidence is presented which suggests that metabolism occurs under both of these conditions. The rate of oxidative metabolism was found to be similar to that of carbon tetrachloride. In anaerobic microsomal preparations fortified with NADPH trichlorofluoromethane exhibited a Soret maximum at 452 nm in the difference spectrum. 3. Experiments in which [[14]C] trichlorofluoromethane was dosed orally to rats support the findings in vitro and show the formation of metabolites in the expired air and urine. The major metabolite was carbon dioxide. 4. [14]C-labelled metabolites of trichlorofluoromethane were found to bind irreversibly to liver microsomal proteins and lipids in vitro. The possibility of similar reactions occurring in vivo is discussed. 5. Evidence is presented which indicates the involvement of the hepatic microsomal mixed function oxidase system in the metabolism of trichlorofluoromethane. In this respect trichlorofluoromethane is very similar to carbon tetrachloride but is not hepatotoxic. This phenomenon is discussed in relation to the possible mechanisms of halogenated hydrocarbon hepatotoxicity.