Search

Acidic glycoconjugates represent the major non-fibrous macromolecular components that form the extracellular and cell-associated matrices of all animal tissues. The constituent molecules are principally structural glycoproteins and proteoglycans. While their protein component is determined by gene pools, it is the polyanionic (acidic) nature of the polysaccharides, determined by their degrees of carboxylation and sulphation, which confers both functional and diagnostic status on these molecules. Sulphated glycoconjugates in the basal laminae have been reported to play a role in tumour invasion and metastasis. In this study, we used cationic colloidal gold together with transmission electron microscopic methods to compare the expression of acidic glyconconjugates in the basal lamina of both normal rat tongue mucosa and experimentally induced oral carcinomas. Results indicated that heparan sulphate rich glycoconjugates were predominant and were mostly confined to the lamina lucida of the basal lamina in normal oral mucosa. Conversely, observation of basal laminae associated with induced carcinomas showed less intense and more widely dispersed gold labelling for heparan sulphate. The observed differences in gold labelling may reflect modified metabolism of sulphated glycoconjugates or result from the action of degradative enzymes in the induced tumours.

Summary An electron microscopical examination of normal rat lingual mucosa and 4-nitroquinoline 1-oxide (4NQO)-induced tongue carcinomas was undertaken. In normal rat tongue, the epithelium of papillae and interpapillary regions exhibited two distinct keratohyalin granule types and essentially similar ultrastructural cellular features in the different epithelial compartments. The interface between epithelium and connective tissue showed a continuous basal lamina. Compared with normal rat tongue epithelium, 4NQO-induced oral carcinomas revealed cellular and nuclear pleomorphism, atypical tonofilament aggregates, increased and swollen mitochondria, dilated intercellular spaces, local discontinuities and thickening of the basal lamina.

There is some evidence that the epithelial cell rests of Malassez partition the root surface from the periodontal ligament blood vessels, and may protect the root from resorption.The aim of the present study was to determine the distributions of the epithelial rests of Malassez (ERM) and blood vessels in the periodontal ligament (PDL) of the developing rat first molar before, during and after emergence.Four Sprague-Dawley rats were sacrificed at two days, one week, two weeks, three weeks, four weeks and six weeks of age. After processing, the maxillae were embedded in paraffin, and sectioned longitudinally and transversely. The sections were stained with a double immuno-histochemical technique which utilised a keratin antibody AE1-AE3 (1:2,000) and an endothelial antibody Factor VIII (1:10,000) to enable simultaneous labelling of ERM and blood vessels. ERM and blood vessel counts were obtained from the mesio-buccal roots of three week, four week and six week-old rats, whilst qualitative observations were made for the earlier developmental stages.ERM cells and cell clusters were found in the tooth third of the PDL width at the three, four and six week stages. Cells and cell clusters increased in number with age, especially in the upper third of the mesio-buccal root. The largest numbers of cells and clusters were found on the distal surfaces of the roots in all age groups. Cells and clusters in all root surfaces increased from three to four weeks, but decreased from four to six weeks. The greatest number of blood vessels was found in the bone-side third of the PDL. The distal surface had the highest proportion of blood vessels, and the palatal surface the least proportion. The number of blood vessels in all surface quadrants did not vary much from three to four weeks of age, but increased from four to six weeks of age, possibly as a reaction to tooth emergence and occlusal function. Physiological root resorption was only observed after tooth emergence, and appeared to be related to loss of continuity of the ERM network and the incursion of blood vessels.Orthodontic root resorption can be regarded as an exaggerated response to loss of PDL homeostatic control, possibly mediated by the epithelial rests of Malassez.

Syndecans are a family of heparan sulphate proteoglycans that regulate cell-matrix interactions that influence cell growth, proliferation and morphology. The aim of this study was to observe changes in the expression of Syndecan-1 in the developing epithelium of the rat oral mucosa and in the epithelial cell rests of Malassez in the developing periodontium of normal rat molars, from late crown development through to early eruption. Immuno-histochemistry (Syndecan-1 N-18) and histochemistry (Alcec Bluel were used to observe changes in the expression of Syndecon-1 in rats aged two to 42 days. Results indicated that during normal tooth development in the rat, labelling or staining of variable intensity for Syndecan-1 was demonstrated in the stratified oral epithelium above the stratum basale in the rat tongue and palate, and in ameloblasts of the developing molar in rats aged two to 14 days. Histochemical staining of the predentine and dentine layers was consistent in all specimens. Labelling or staining for Syndecan-1 was negative in the rat periodontal ligament, which may suggest that either Syndecan-1 was not expressed during normal molar root development or that continued work is required for identification of a suitable label in rats.

Invasive growth of cancer cells is a complex process involving specific interactions between tumour cells and the orderly, integrated complexes of the extracellular matrix. Basement membranes have been proposed as one constituent of extracellular matrix which carries responsibility for regulating invasion and metastasis. Using a chemically induced rat tongue carcinoma model, it has been shown that components of the basement membrane and its overall structure are altered during tumour invasion, and methods have been developed to quantitate some of these differences. Since the basement membrane can be specifically characterized by its fibrous protein network of Type IV collagen and laminin, which is embedded in a heparan sulphate-rich proteoglycan matrix, these components have been targeted. In particular, the current paper presents results in the context of current concepts of early changes in neoplastic invasion of underlying connective tissues. In consequence, further elaboration of the underlying mechanisms of epithelial migration in oral cancer may allow an exploration of the use of alterations in expression of basement membrane components as prognostic indicators.

The purpose of this study was to undertake preliminary analyses of the extracellular proteoglycans in carcinogen [4-nitroquinoline N-oxide (4NQO)]-treated rat tongue mucosa. Experimental rats were exposed to twice-weekly applications of 4NQO in propylene glycol for six months, after which the animals were killed. Control and 4NQO-treated tissues were subjected to sequential aqueous extractions of proteoglycans under associative and dissociative conditions, followed by alkaline cleavage of protein-glycosaminoglycan linkages to yield a glycosaminoglycan residue. Tissues subjected to 4NQO applications contained smaller proportions of proteoglycans which were readily soluble under associative and dissociative conditions. Proportionately more proteoglycan remained strongly associated with other intercellular tissue components, being released only by alkaline cleavage. These biochemical alterations in preinvasive 4NQO-treated epithelium and connective tissues, together with an observed associated change in water retention by the connective tissue, occurred prior to actual neoplastic invasion and suggest differences in macromolecular conformation and orderliness. We hypothesize that these changes are related to the phenomenon of neoplastic epithelial invasion.

Glutathione (GSH) is an endogenous thiol that detoxifies active oxygen and reactive species formed during intermediary metabolism and drug detoxification. Compounds with a range of potential toxicities were; tested for their abilities to affect GSH reductase and GSH S -transferase activities, which are each components of the two principal detoxification pathways in which GSH participates. A high performance liquid chromatographic method for determining oxidized and reduced GSH was modified to assay GSH reductase activity. With this method it was possible to demonstrate that ethacrynic acid, which inhibits GSH S -transferase, also inhibits the activity of GSH reductase. Inhibition of GSH reductase by ethacrynic acid was similar to that seen with carmustine (BCNU). GSH reductase activity was not affected by cis- or transplatin, buthionine sulfoximine, other loop diuretics, cyclosporine A or aminoglycosides. Cyclosporine inhibited GSH S -transferase at 50 μM and higher concentrations. These results support a role for GSH-mediated detoxification mechanisms in ethacrynic acid- and cyclosporine-associated cytotoxicity, which may mediate their toxicities and their potential as adjunctive agents in antineoplastic therapy. A better understanding of the mechanism of their toxicity can greatly extend the clinical usefulness of these agents, as this toxicity is the basis of both their therapeutic and antitherapeutic actions.

The relationship of basal lamina, a form of specialised extracellular matrix which separates epithelial cells and other cell types from adjacent stroma, to the behaviour of malignant neoplasms of epithelial origin is not well understood. However, it is widely acknowledged that the properties of local invasion and metastasis of carcinomas are linked to extracellular matrix (including basal lamina) changes. In the present study, the distribution of the major basal lamina components, type IV collagen and laminin, in normal rat tongue mucosa and experimentally induced oral carcinomas was investigated using post-embedding immunogold techniques and electron microscopy. The expression of these components was also quantitatively analysed using morphometry and immunocytochemistry. Results indicated that type IV collagen and laminin were confined to the lamina densa of normal oral epithelial basal lamina, and that both components were also detected in the lamina densa of basal lamina associated with carcinomas, and in the extracellular matrix of tumours. Furthermore, laminin was detected within stromal fibroblasts in normal tissues and experimental carcinomas. Quantitative analysis indicated that expression of laminin was significantly increased in carcinomas. In contrast, type IV collagen expression was significantly decreased. The quantitative changes observed in the two basal lamina constituents may be related to the process of tumour invasion, reflecting altered metabolic activities of tumour and stromal cells. These observations may be of use in understanding the architectural characteristics of oral mucosa basal lamina and in assessing the malignant potential of epithelial dysplasias or "premalignant" lesions.

Summary The pattern of staining and the distribution of laminin and type IV collagen in normal rat tongue mucosa and induced tongue carcinomas were investigated by immunohistochemical techniques. Both normal and neoplastic epithelial basement membrane revealed positive staining for laminin and type IV collagen. However, compared with normal tissue, carcinomas exhibited areas of increased density and thickness for laminin. Focal tumour basement membrane discontinuities were observed in some specimens stained for type IV collagen.

The biochemical and histochemical measurement of the enzyme γ-glutamyltransferase (GGT) was undertaken in normal rat pancreas and in rat pancreas containing azaserine-induced preneoplastic nodules. A steady decrease in pancreatic GGT activity was observed in the normal animals as they aged from 5 to 34 weeks. The azaserine-induced nodules contained a lower average GGT activity than the control pancreas although a 10-fold variation was noted in the GGT activity of individual nodules. A significant increase in concentrations of both reduced glutathione and oxidized glutathione was noted in pancreatic nodules from azaserine-treated rats compared to concentrations found in both control pancreas from untreated rats and internodular pancreas from azaserine-treated rats. A pancreatic acinar cell carcinoma contained low GGT activity-similar to that found in large nodules and about 10% of the level found in control pancreas. Pancreatic GGT levels were higher in 5- and 7-week-old rats fed chow than in rats fed a purified diet, but this effect of chow was not observed at 34 weeks of age. Feeding a purified diet supplemented with a retinoid, N -2-hydroxyethylretinamide (2-HER), for a period of 2 weeks did not influence the GGT activity level in either normal pancreas or in the azaserine-induced nodules. While decreased GGT activity does not serve as a marker for all atypical acinar cell nodules, deficient activity with concomitant increased glutathione levels appears to correlate generally with increased growth potential.

The metabolism and toxicity of some ethyl-substituted organotin compounds in isolated rat hepatocytes were studied. Tetra- and triethyltin derivatives were metabolized by isolated rat hepatocytes to yield ethane and ethylene. Hydrocarbon formation from tetraethyltin was larger than that obtained with triethyltin bromide, and ethylene was the major product (95%) of tetraethyltin metabolism. At a triethyltin salt concentration of 100 μM, the major product formed by cells from untreated rats was ethane. Pretreatment in vivo by phenobarbital resulted in a marked increase in the overall rate of hydrocarbon production and a change in ethylene to ethane ratio; in this case ethylene was the predominant metabolite produced. 5,6-Benzoflavone pretreatment in vivo resulted in a small depression in overall hydrocarbon production. No metabolism of diethyltin dichloride (100 μM) by isolated rat heptocytes was detected. Triethyltin bromide (100 μM) was a potent inhibitor of both the Phase I (oxidation) and Phase II (conjugation) metabolism of biphenyl in isolated hepatocytes from phenobarbital-pretreated rats, whereas diethyltin dichloride was seen to affect particularly the Phase I metabolism of the aromatic hydrocarbon. Triethyl- and diethyltin salts reduced oxygen consumption and ATP levels in these cells. However, the triethyl derivative was more effective in this respect. Tetraethyltin was not appreciably toxic to hepatocytes. Trypan blue dye exclusion and lactate dehydrogenase loss by the cells isolated from phenobarbital-pretreated rats indicated that triethyltin bromide was more toxic than diethyltin dichloride. In contrast, the diethyl derivative was more potent in stimulating lipid peroxidation as indicated by formation of thiobarbituric acid-reactive products than was triethyltin.

Surgicel, a local haemostatic gauze, is claimed to consist of oxidised regenerated cellulose. It is a polyanion, the functional unit of which is termed polyanhydroglucuronic acid. The ability of tissues to absorb Surgicel and its inherent haemostatic properties have been extensively investigated. This study was undertaken a) to determine the time required for absorption of Surgicel from implantation sites in the chest wall muscles of rats, and b) to establish mechanisms for its removal. Data derived from sequential uronic acid assays, histochemistry using the stain alcian blue, and transmission electron microscopy of implanted Surgicel were interpreted to reveal that Surgicel consists of at least two active components. These are a soluble uronic acid component which is lost after 6 h, and a fibrous component which persists. The latter material resembles Surgicel in the electron microscope and is still evident at the implantation site at 48 h post-implantation. Moreover, Surgicel can be characterized in vitro into at least two components according to its solubility under dissociative salt conditions (4M guanidinium chloride). A residual fibrous material could then be hydrolysed with 0.3N sodium hydroxide. We postulate that the absorption of the former salt soluble uronate in vivo is by early degradation and/or systemic clearance, whilst removal of the fibrous material requires phagocytosis.

Previous reports have demonstrated that Surgicel ® , a local haemostatic agent, is absorbed from implantation sites. In an earlier study, it was shown that the material consists of a uronic acid component and a fibrous residue. A chemically quantified loss of the uronic acid component within 18 h of implantation was demonstrated. The aim of the present study was to examine the fate of the fibrous residue in rat tissues, using both light and electron microscopy. Results indicated that this fibrous component is phagocytosed by macrophages at the site of implantation. A model for the clearance of Surgicel from tissue implantation sites is presented.

The NADPH- and oxygen-dependent microsomal metabolism of the di-, tri- and tetra-ethyl-substituted derivatives of germanium, tin and lead was shown to give rise to ethylene as a major product and ethane as a minor product. These reactions were shown to be catalysed by the liver microsomal cytochrome P-450-dependent mono-oxygenase. Since formation of ethane and ethylene was differentially inhibited by anaerobiosis, the results suggest that at least a large portion of the ethane produced may be derived by a reductive mechanism. Triethyltin bromide in both the absence and presence of NADPH was shown to convert cytochrome P-450 into cytochrome P-420 and to affect the function of the mono-oxygenase in vitro. Tetraethyltin caused the NADPH- and time-dependent formation of cytochrome P-420, suggesting that tetraethyltin is converted into triethyltin salts in significant concentrations. The order of potency in formation of cytochrome P-420 was closely paralleled by the ability of the tin derivatives to induce microsomal lipid peroxidation in vitro.

1. The metabolism of biphenyl by isolated viable rat hepatocytes has been studied and a tentative scheme of metabolism proposed which involves initial hydroxylation at the 2- and 4-positions followed by conjugation and/or further hydroxylation of these primary metabolites. 2. Biphenyl was toxic to viable hepatocytes when used at a concentration approaching that used in conventional microsomal assay systems. 3. The production of small amounts of 4-hydroxybiphenyl appears to activate its subsequent conjugation. 4. The data presented in this paper integrate previous results obtained with cell fractions, and demonstrates the importance of the isolated, viable hepatocyte system as a model for total drug metabolism.

Cyclophosphamide (CPA), a compound requiring metabolic activation to form toxic metabolites, has been used as a model compound to validate the use of a hepatocyte/mouse mastocytoma (line P815) cell culture system capable of measuring metabolism-mediated cytotoxicity in vitro . A number of hydrazines were tested in this system and were found to be relatively non-toxic. However, an attempt has been made to correlate complex formation between hydrazine metabolites and cytochrome P-450 in rat hepatocytes and the inhibition of the metabolism-mediated cytotoxicity of CPA. N -Aminopiperidine (NAP) was the most potent hydrazine tested in this system, since it significantly reduced the metabolism-mediated toxicity of CPA. The concentration dependency of this phenomenon permitted the calculation of the amount of NAP that reduced CPA cytotoxicity by 50%. This value (approximately 1 mM) correlated well with the apparent Michaelis constant (1.2 mM) for the formation of the inhibitory metabolite complex with cytochrome P-450 in microsomal suspensions. There was good correlation between the abilities of a number of 1,1-disubstituted hydrazines to reduce the metabolism-mediated toxicity of CPA and the extent to which they form the inhibitory metabolite complex in microsomes. 1,2-Dimethylhydrazine did not form an observable inhibitory complex in microsomal suspensions and only slightly reduced the metabolism-mediated toxicity of CPA in the mouse mastocytoma cell culture system. However, some other hydrazine derivatives were shown to alter significantly cytochrome P-450 function in isolated hepatocytes. This in vitro system may be of use in the evaluation of the biological effects of xenobiotics, particularly those requiring metabolic activation by cytochrome P-450.

The microsomal monooxygenase system of adult rat hepatocytes in short-term non-proliferating culture could be induced by phenobarbitone and benzanthracene. Differences in the kinetics of induction and the additive nature of the inductions indicated that induction by the two agents occurred by different mechanisms and the use of haemoprotein-selective inhibitors demonstrated the induction of different haemoproteins by the two agents. The type of haemoprotein present in the cells altered during a four day culture period.

Two 2-aminofluorene-derived hydroxamic acids that differ only in the nature of the N-acyl group were examined for their relative abilities to undergo covalent binding to nucleic acids. Studies of the bioactivation of N-hydroxy-N-acetyl-2-aminofluorene (N-OH-AAF) and N-hydroxy-N-glycolyl-2-aminofluorene (N-OH-GAF) were conducted with hepatocyte suspensions and subcellular fractions prepared from male Sprague-Dawley rats. Both hydroxamic acid substrates displayed equal binding to both DNA and RNA after incubations with hepatocyte suspensions. The extent of binding of each substrate was approximately the same for DNA and RNA. Investigations with subcellular fractions revealed some major differences between the probable mechanisms by which the two substrates were covalently bound to exogenous DNA. In agreement with the prior literature reports, N-OH-AAF was extensively bound to DNA through the action of cytosol enzymes, including both N,O-acyltransferase and sulfotransferase. The microsomal enzyme fraction also catalyzed binding to DNA, and this process was completely inhibited by paraoxon. The covalent binding of N-OH-GAF to DNA was catalyzed by cytosol enzymes to a significant extent only in the presence of 3'-phosphoadenosine-5'-phosphosulfate, which suggests the action of sulfotransferase. Covalent binding of N-OH-GAF to DNA was minimal through the action of cytosolic N,O-acyltransferase, which confirms our earlier observation that N-OH-GAF is a potent suicide inhibitor of this enzyme. The microsomal fraction catalyzed the binding of N-OH-GAF to DNA at a rate that was about twice that observed for N-OH-AAF.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolated rat and hamster acinar cell suspensions possess the ability to carry out the cytochrome P-450-dependent O-deethylation of 7-ethoxycoumarin, 2-,3-, and 4-hydroxylation of biphenyl and 3-hydroxylation of benzo(a)pyrene. Rat and hamster acinar cells isolated from 5,6-benzoflavone-pretreated animals oxidize all three substrates at measurable rates. These rates are considerably lower (16-210-fold in the rat and 290-2670-fold in the hamster) than those in incubations using hepatocytes isolated from 5,6-benzoflavone-pretreated animals. Hydroxylation of biphenyl at the 2-, 3-, and 4-positions proceeds at similar rates in rat acinar cells. The rate of 3-hydroxybiphenyl formation is barely detectable in hamster acinar cells where the rates of 2- and 4-hydroxybiphenyl formation are slower than in the rat. No detectable oxidation products of 7-ethoxycoumarin, biphenyl, or benzo(a)pyrene are found with acinar cells of either species isolated from untreated and phenobarbital-pretreated animals. The O-deethylation of 7-ethoxycoumarin in rat and hamster acinar cells is decreased in the presence of inhibitors of the cytochrome P-450-dependent monooxygenase system, 7,8-benzoflavone being much more effective than metyrapone. The deethylation product of 7-ethoxycoumarin, 7-hydroxycoumarin, is conjugated with sulfate and glucuronic acid moieties at appreciable rates by acinar cells isolated from both rat and hamster. Pretreatment of rats and hamsters with either 5,6-benzoflavone or phenobarbital has little effect on the rates of conjugation in isolated acinar cell preparations.

Utilizing the technique of alkaline elution analysis, the ability of N-nitrosomethyl(2-oxopropyl)amine (MOP), a potent pancreatic carcinogen, to damage pancreatic DNA in rats and hamsters was examined. Pancreatic DNA isolated from hamsters exposed for 1 h to MOP given i.p. at doses of 7-60 mg/kg showed dose-related DNA damage. A similar dose-response was observed in the pancreas of rats receiving 20-180 mg MOP/kg, suggesting that hamsters were 2-3 times more sensitive than rats. In contrast to the results obtained in vivo, functionally viable acinar cells from both rat and hamster pancreas, when exposed in vitro to levels of MOP comparable to those in vivo (20-180 micrograms/ml), failed to show dose-related DNA damage. Acinar cells from hamsters pretreated with 5,6-benzoflavone, an inducer of cytochrome P-450 activity, showed greatly enhanced drug-metabolizing capability, but again no DNA damage was observed upon exposure to MOP. Minced hamster or rat pancreas also failed to show DNA damage in response to MOP treatment. When hamsters in which hepatic blood supply was interrupted by ligation were given 60 mg/kg MOP i.v. and sacrificed 15 min later, damage to pancreatic and liver DNA was comparable to that observed in ligated controls which had received saline only. Administration of MOP to sham-operated animals led to extensive DNA damage in both pancreas and liver at 15 min. Analysis by h.p.l.c. showed an almost 2-fold increase in the amount of MOP present in the pancreases of the liver-ligated animals as compared to the sham-operated unligated animals. MOP was absent from the liver of the ligated animals. These experiments strongly suggest that DNA damage by MOP to the pancreatic acinar cells and probably to other pancreatic cell types, as well, requires metabolic activation by the liver.

Biphenyl is metabolised by untreated isolated rat hepatocytes via hydroxylation at the 4-position followed by conjugation with sulphate and glucuronic acid. At a substrate concentration of 70 μM, the major metabolite produced is 4-hydroxybiphenyl sulphate. Hydroxylation at the 2- and 3-positions also occurs but to a much smaller extent. Induction in vivo by phenobarbitone or 3-methylcholanthrene results in a marked increase in the rate of the initial hydroxylation and a change in the pattern of the conjugates with 70 μM biphenyl. In the induced state 4-hydroxybiphenyl glucuronide is the dominant metabolite, 4-hydroxybiphenyl sulphate production being similar to controls. Two- and 3-hydroxylation of biphenyl in isolated rat hepatocytes is markedly increased by 3-methylcholanthrene pretreatment, but is unaffected by phenobarbitone pretreatment. Studies on the metabolism of two of the primary metabolites of biphenyl, 2- and 4-hydroxybiphenyl, in untreated isolated rat hepatocyte suspensions revealed that whereas at low (7 μM) concentrations of both metabolites, sulphate conjugation was predominant, with increasing substrate concentration the contribution of the glucuronidation pathway was elevated to such an extent that it became the predominant conjugating pathway at the highest concentrations used (140 μM). The reasons for the lag between the ‘initial hydroxylation’ and the onset of glucuronic acid conjugation of 4-hydroxybiphenyl were investigated. Attempts were made to eliminate this lag by prior incubations of the cells with 4-methylumbelliferone in an effort to activate the conjugating enzymes responsible. Glucuronidation of 4-hydroxybiphenyl is activated significantly by this treatment, whereas sulphation of 4-hydroxybiphenyl is not.

The effects of exposure of adult rat hepatocytes to chemical carcinogens have been studied using a short-term maintenance culture system. Scanning microdensitometry was used to quantitate the observed changes in enzyme activity. The dose-response curves showed a biphasic response for all 4 enzymes studied (glucose-6-phosphate dehydrogenase, succinate dehydrogenase, NADPH oxidase and gamma-glutamyl transpeptidase) there being decreased enzyme activities at the higher dose levels used, possibly indicating cytotoxicity. The enhancement of enzyme activity at low dose levels was due to generalised increases occurring in every cell, rather than to selection of a cell species particularly high in enzyme activity. A culture period of 24 h was necessary for the complete adaptation of the cells to the culture environment as evidenced by the response of intracellular glucose-6-phosphate dehydrogenase activity to carcinogen treatment. These findings are discussed in relation to previously reported in vivo studies.

1. The metabolism of a number of xenobiotics has been investigated in isolated viable rat hepatocytes and kidney tubule fragments in suspension. 2. The level of Phase I metabolism was very low in the kidney cells although such cells possess appreciable Phase II metabolic capacity. 3. These findings are discussed in relation to the physiological role of cytochrome P-450 and the conjugating enzymes, and the value of intact cells systems in assessing inter-organ differences in xenobiotic metabolism.

The NADPH-dependent microsomal metabolism of [14C]procarbazine, labeled on the terminal N-methyl group, resulted in the covalent binding of the drug to exogenously added DNA; this reaction was inhibited by metyrapone. Procarbazine metabolism was also shown to result in covalent binding of the methyl group of the drug to microsomal protein upon metabolism, but the extent of protein binding was at least an order of magnitude smaller than that seen with its primary oxidative metabolite. N-isopropyl-alpha-(2-methylazo)-p-toluamide. The characteristics of the reactions leading to the covalent binding of the N-methyl group of the azo derivative to microsomal protein and its metabolism to form the hydrocarbon, methane, possessed a number of similarities in the apparent kinetic parameters (Km and Vmax), induction, and inhibition patterns indicating a common pathway of metabolism to form a reactive intermediate and the involvement of cytochrome P-450. Reduced glutathione stimulated methane formation and inhibited covalent binding to protein. One azoxy derivative, N-isopropyl-alpha-(2-methyl-ONN-azoxy)-p-toluamide, was chemically unstable and its decomposition was shown to lead to covalent binding to microsomal protein. A diazene intermediate and a methyl radical are proposed to be intermediates in the formation of methane during the oxidative metabolism of the azo derivative of procarbazine and a common intermediate in the activation of procarbazine may result in both covalent binding to cellular macromolecules and methane production. In addition, chemical decomposition of the azoxy metabolites may also contribute to a small portion of the covalent binding, but not to methane formation.

Various methods for the preparation of rat hepatocyte suspensions have been compared with regard to cell yield and viability index. A modified method of collagenase/hyaluronidase digestion using a sequential removal and replacement of Ca 2+ and the omission of all perfusion steps has been evolved, thereby making the technique potentially viable for use with pieces of liver such as isolated human liver biopsy material. These suspensions contain large numbers of viable cells, and the viability index is also high (>70%). A number of metabolic functions can be readily detected in these cell suspensions, indicating the intactness of the isolated cells. A systematic survey of the enzymic components required for liver digestion is also reported.

The effect of three inhibitors of mitochondrial function (menadione, rotenone and 2,4-dinitrophenol) on drug metabolism in isolated rat hepatocytes has been studied. Menadione (at 1.25 × 10−4 M) caused almost complete inhibition of biphenyl Phase I metabolism whereas rotenone (2 × 10−5 M) inhibited the same reaction only by 25 per cent although the subsequent conjugation of the Phase I metabolite was markedly depressed. Qualitatively similar findings were observed with hepatocytes isolated from phenobarbital-pretreated rats, and with liver microsomes isolated from control rats. 2,4-Dinitrophenol (2 × 10−4 M) caused a marked enhancement of biphenyl Phase I metabolism but a marked inhibition of subsequent conjugation. This enhancement of Phase I metabolism was not observed in control cells with other substrates (benzo[a]pyrene, 7-ethoxycoumarin), nor in biphenyl metabolism in “induced” cells or in liver microsomes isolated from control rats. It is tentatively suggested that products of 2,4-dinitrophenol metabolism may “activate” biphenyl metabolism in intact liver cells. Furthermore, it is suggested for all three inhibitors that direct effects on the drug metabolizing enzyme systems (Phase I and Phase II) are as important as their effects on mitochondrial function in explaining their inhibition of drug metabolism. It appears that Phase II metabolism of xenobiotics is more susceptible to inhibition by metabolic inhibitors than is Phase I metabolism, probably due to depletion of the cellular ATP levels.