Metabolic Competence Research Articles

Phase 1 and 2 Metabolism in Freshly Isolated Hepatocytes and Subcellular Fractions from Rat, Mouse, Chicken and Ox Livers

In toxicological studies hepatocytes offer an excellent alternative to whole-animal experiments, provided their metabolic competence has been established. We have compared Phase 1 and 2 metabolism in rat, mouse, chicken and ox liver microsomes and cytosol with freshly isolated hepatocytes. The relative amounts of total cytochrome P450 in microsomes and hepatocytes were equivalent. Rat liver had the highest P450 content while chicken liver had the lowest content (148·2(±75·7) and 20·6(±11·5) pmol mg-1 hepatocellular protein, respectively). The metabolism of testosterone was assessed to determine selective cytochrome P450 isoenzyme activities. Only two metabolite products were common to all four species, namely 6β-hydroxytestosterone (6β-OHT) and androstenedione (ASD), which co-eluted with 6-dehydrotestosterone (6DHT). 16α-OHT was present in all incubations except for ox microsomes. The rate of metabolism of testosterone was generally lower in microsomes than hepatocytes, with the exception of the ox, but the pattern and quantity of metabolite formation was similar. The quantity of total products formed was 15- to 27-fold higher in rat and mouse livers than in chicken or ox. The major product formed in freshly isolated hepatocytes from mice and chickens was ASD/6DHT which accounted for 60% and 76% of the total metabolites, respectively. ASD/6DHT formation accounted for only 33% and 17% of the total metabolites formed by rat and ox hepatocytes, respectively. 2α-OHT production occurred in rat and mouse hepatocytes (14% of the total metabolites in rat and 7% in mouse hepatocytes) but was lacking in chicken or ox cells. The stability of P450 isoforms in culture was species-dependent. Rat and mouse hepatocyte cultures lost 54% and 31% of their initial P450 content after 72 h, while there was no loss in chicken hepatocytes over the same period. There was a good correlation between the relative glutathione S-transferase (GST) activities in cytosol and freshly isolated hepatocytes. Mouse liver exhibited highest GST activity (664·2(±203·5)) compared with rat, chicken or ox (320·4(±64·0), 341·5(±13·9) and 256·3(±109·9) nmol min-1 mg-1 cytosolic protein, respectively). © 1997 SCI.

Phase 1 and 2 Metabolism in Freshly Isolated Hepatocytes and Subcellular Fractions from Rat, Mouse, Chicken and Ox Livers

In toxicological studies hepatocytes offer an excellent alternative to whole-animal experiments, provided their metabolic competence has been established. We have compared Phase 1 and 2 metabolism in rat, mouse, chicken and ox liver microsomes and cytosol with freshly isolated hepatocytes. The relative amounts of total cytochrome P450 in microsomes and hepatocytes were equivalent. Rat liver had the highest P450 content while chicken liver had the lowest content (148·2(±75·7) and 20·6(±11·5) pmol mg-1 hepatocellular protein, respectively). The metabolism of testosterone was assessed to determine selective cytochrome P450 isoenzyme activities. Only two metabolite products were common to all four species, namely 6β-hydroxytestosterone (6β-OHT) and androstenedione (ASD), which co-eluted with 6-dehydrotestosterone (6DHT). 16α-OHT was present in all incubations except for ox microsomes. The rate of metabolism of testosterone was generally lower in microsomes than hepatocytes, with the exception of the ox, but the pattern and quantity of metabolite formation was similar. The quantity of total products formed was 15- to 27-fold higher in rat and mouse livers than in chicken or ox. The major product formed in freshly isolated hepatocytes from mice and chickens was ASD/6DHT which accounted for 60% and 76% of the total metabolites, respectively. ASD/6DHT formation accounted for only 33% and 17% of the total metabolites formed by rat and ox hepatocytes, respectively. 2α-OHT production occurred in rat and mouse hepatocytes (14% of the total metabolites in rat and 7% in mouse hepatocytes) but was lacking in chicken or ox cells. The stability of P450 isoforms in culture was species-dependent. Rat and mouse hepatocyte cultures lost 54% and 31% of their initial P450 content after 72 h, while there was no loss in chicken hepatocytes over the same period. There was a good correlation between the relative glutathione S-transferase (GST) activities in cytosol and freshly isolated hepatocytes. Mouse liver exhibited highest GST activity (664·2(±203·5)) compared with rat, chicken or ox (320·4(±64·0), 341·5(±13·9) and 256·3(±109·9) nmol min-1 mg-1 cytosolic protein, respectively). © 1997 SCI.

In vivo nitrogen metabolism in ornithine transcarbamylase deficiency.

We developed a new technique that monitors metabolic competency in female heterozygotes for ornithine transcarbamylase deficiency (OTCD). The method uses mass spectrometry to measure conversion of (15)NH4Cl to [15N]urea and [5-(15)N]glutamine following an oral load of (15)NH4Cl. We found that heterozygotes converted significantly less NH3 nitrogen to urea, with this difference being particularly obvious for symptomatic carriers, in whom the blood [15N]urea concentration (mM) was significantly less than control values at most time points. The blood concentration of [5-(15)N]-glutamine (microM) was significantly higher in both asymptomatic and symptomatic heterozygotes than it was in the control subjects. The administration of a test dose of sodium phenylbutyrate to the control group did not affect the rate of [15N]urea formation. We conclude: (a) This test effectively monitors in vivo N metabolism and might obviate the need for liver biopsy to measure enzyme activity in OTCD; (b) Asymptomatic OTCD carriers form urea at a normal rate, indicating that ureagenesis can be competent even though enzyme activity is below normal; (c) Although ostensibly asymptomatic OTCD carriers form urea at a normal rate, their nitrogen metabolism is still abnormal, as reflected in their increased production of [5-(15)N]glutamine; and (d) This new test may be important for monitoring the efficacy of novel treatments for OTCD, e.g., liver transplantation and gene therapy.

Age-dependent decrease in the activity of succinic dehydrogenase in rat CA1 pyramidal cells: a quantitative cytochemical study

A computer-assisted morphometric study has been carried out on the ultrastructure of perikaryal CA1 pyramidal cell mitochondria positive to the copper ferricyanide cytochemical reaction for succinic dehydrogenase (SDH) in rats of 3, 12 and 23 months of age. The cytoplasmic volume fraction occupied by the positive mitochondria (Volume density: V v), the number of organelles/μm 3 of CA1 pyramidal cell cytoplasm (Numerical density: N v) and the average mitochondrial volume ( V) were automatically calculated by means of computer-assisted morphometry. V v was significantly decreased in 23-month-old animals versus the other age groups. N v was unchanged between 3 and 12 months of age, but was decreased to a significant extent in old animals. V did not undergo significant changes in the three age groups taken into account. In the old animals the percent of organelles smaller than 0.16 μm 3 is above 20%, while in the young and adult groups the same size of mitochondria accounts for 7 and 3%, respectively. Thus, a reduction in the number of medium sized organelles appears to be responsible for the decrease in V v due to age. Since SDH activity is known to support maximum rates of respiration, quantitative estimation of the active mitochondria provides information on the metabolic competence of the cells investigated when energy demand is high. In this context, our present findings document that a significant impairment in the efficiency to match actual energy provisions occurs in old CA1 pyramidal cells.

Oil palm (Elaeis guineensis) protoplasts: isolation, culture and microcallus formation

Procedures are deseribed for the efficient isolation of protoplasts from a variety of oil palm (Elaeis guineensis Jacq.) tissues. Various factors including donor source, composition of enzyme mixture and culture medium affected the yield and viability of the protoplasts Polyembryogenic cultures of oil palm were the most suitable starting material in terms of yield, viability and metabolic competence. Pectolyase Y-23 in association with cellulase and hemicellulase was required for the efficient release of protoplasts from the oil palm tissues. Limited cell division to form microcallus was observed at very low frequency (<0.01%) when glutathione and catalase were incorporated in the culture medium.

Erythrocyte enzymes catalyze 1-nitropyrene and 3-nitrofluoranthene nitroreduction

Nitroarenes are environmental contaminants produced during incomplete combustion processes. Nitroreduction, the most important pathway of nitroarene toxification, occurs mainly in the liver and intestine. In the present study, we show that human red cells may also possess the metabolic competence to reduce 1-nitropyrene (NP) and 3-nitrofluoranthene (NF), the nitroarenes chosen as model compounds, to their corresponding amino derivatives, 1-aminopyrene (AP) and 3-aminofluoranthene (AF). The requirement of the cofactor couple NADH/FMN suggests that erythrocyte nitroreductase activity occurs via one electron transfer. The presence of oxygen strongly inhibited the haemolysate-catalyzed nitroarene reduction, whether measured as amine formation or nitroarene disappearance. Intermediate reactive species, that bind covalently to haemoglobin and/or other erythrocyte proteins, are formed during nitroreduction catalyzed by human haemolysate. In fact, the reduced metabolites AP and AF were released after mild acid hydrolysis of red cell proteins exposed to NP and NF, thus suggesting that sulphinamide adducts have been formed.

Metabolic activation of benzo[ a]pyrene in two fetal mouse hepatocyte lines: induction of DNA adducts and micronuclei

We have studied the metabolic competence of two non-transformed epithelial-like cell lines derived from fetal mouse liver, C 6 and C 2.8, to activate the promutagen benzo[ a]pyrene by measuring both the induction of DNA adducts through the nuclease P1-enhanced 32P-postlabeling assay and the formation of micronuclei. The pattern and level of DNA adducts detected in C 6 and C 2.8 cells treated with benzo[ a]pyrene were compared with those obtained in human peripheral blood lymphocytes treated with the same compound and with [ 3H]anti-benzo[ a]pyrene diolepoxide. In both the cell lines and in human lymphocytes we observed a consistent induction of distinct DNA adducts. In C 6 and C 2.8 cells, the most evident adduct showed a position similar to that of the main adduct induced by [ 3H]-anti-benzo [a] pyrene diolepoxide in human lymphocytes. In addition, benzo[ a]pyrene caused a significant increase of micronucleated C 6 and C 2.8 cells, whereas the frequency of micronuclei did not increase in CHO cells treated, for comparison, in the same way.

Assessment of embryo potential by visual and metabolic evaluation

Morphological evaluation of embryos is essential to the success of embryo transfer procedures and is presumed to reflect embryo metabolic activity. To investigate this assumption, correlations between morphological and metabolic parameters were determined for cultured murine morulae. After 18 h (n = 47) or 36 h (n = 48) of culture in M16, the developmental rate and quality (poor or good) of embryos were estimated, and, then, either their 14c-glucose utilization or 35s-methionine uptake and incorporation were measured. Retarded developing, or poor-quality embryos had lower mean glucose utilization, uptake and incorporation rates than normally developing or good-quality embryos (P < 0.05). After 18 h of culture, an association was found between developmental rate and metabolic activity, but this was not evident after 36 h of culture. Similarly, an association was found between embryo quality and metabolic activity. As expected, poor embryo quality was indicative of low metabolism throughout the culture period, but good quality did not necessarily indicate normal metabolic activity. Thus, morphological parameters do not always reflect metabolic competence, and some functional defects were not detectable by visual evaluation alone. Measuring metabolic parameters could complement visual evaluation for a better selection of embryos prior to transfer.

In vivo and in vitro efficacy of a new carbodithioate for the mobilization of cadmium.

Female Wistar rats with chronic cadmium intoxication (oral exposure to low dosages of CdCl2 in drinking water over a period of 90 d) were used to examine the in vivo ability of a newly developed chelator, sodium N-(4-methylbenzyl)-4-O-(beta-D-galactopyranosyl)-D-glucamine-N- carbodithioate (MeBLDTC), singly and in combination with sodium 4-carboxy-amidopiperidine-N-carbodithioate (INADTC) as agents to induce the biliary and urinary excretion of cadmium. The combined administration of the two dithiocarbamates, which differ greatly in molecular weight and structural features, led to a synergistic increase in the biliary excretion of cadmium and an enhanced reduction of renal cadmium levels. The use of such a coadministration produced an increase in the biliary excretion of cadmium that was more than double that expected if the compounds acted in an additive fashion. Such mixed-chelation therapy has potential utility in the treatment of human chronic cadmium intoxication. The hepatocytes isolated from chronically Cd-intoxicated rats were used as an in vitro screening model system for the new chelator. The plasma membrane integrity study with MeBLDTC at 0.48 mmol/20 ml of hepatocyte incubate using the trypan blue exclusion test and lactate dehydrogenase (LDH) leakage test revealed no differences in the cell viability with or without chelator. The cellular metabolic competence measured as the rate of urea synthesis also did not show any marked deviation from that of controls when incubated with MeBLDTC at three different concentrations. In the hepatocyte cultures, MeBLDTC induced a significant removal of cadmium from the hepatocytes at concentrations as low as 0.04 mmol/20 ml.

Sexual selection and the energetics of copulatory courtship in the Sierra dome spider, Linyphia litigiosa

Abstract. Mating in the Sierra dome spider, Linyphia litigiosa (Linyphiidae) begins with 2-6 h of aspermic copulatory courtship, termed pre-insemination phase copulation. Male intromission rate, that is, the speed of repeated genitalic insertions, withdrawals and re-insertions, and body mass are related to fertilization success via cryptic female choice (i.e. female preferences are expressed in their sperm use patterns; Watson 1991b, Anim. Behav., 41, 343-360). Carbon dioxide (CO2) respirometry indicated that pre-insemination phase copulation is energetically demanding. Male intromission rate and body mass (which are uncorrelated traits) each were positively associated with different subsets of six complementary measures of metabolic competence made during copulatory courtship; (1) efficiency; (2, 3) maximum metabolic rate and scope; (4, 5) rate and linearity of metabolic increase; (6) the consistency of metabolic output during sustained courtship. Composite ranks of males based on the six metabolic and the two overt phenotypic measures of quality were positively correlated. Body mass and intromission rate also were associated with two aspects of male fighting ability, strength and aggressiveness (i.e. willingness to escalate when at a modest size disadvantage), respectively. Fighting is important for defence of webs, prey and mates. Thus metabolic competence, a fundamental and possibly heritable component of viability, is favoured by sexual selection; this may help to yield offspring that fare better under both natural and sexual selection.

Cultured trout liver cells: utilization of substrates and response to hormones.

The characterization of a recently established system for the short-term culture of rainbow trout (Oncorhynchus mykiss) liver cells in chemically defined medium has been extended to studies on the metabolic competence of the cells and the characterization of their response to hormones. Three areas of metabolism have been addressed: a) the utilization of the exogenously added substrates fructose, lactate, glucose, dihydroxyacetone, and glycerol for glucose and lactate formation; b) the effects of the pancreatic hormones insulin and glucagon on cellular glucose formation, lactate formation, and fatty acid synthesis; and c) the effects of insulin and dexamethasone on the estradiol-dependent production of vitellogenin. Incubation of trout liver cells with fructose, lactate, glucose, dihydroxyacetone, or glycerol resulted in enhanced rates of cellular glucose and lactate production. Substrate-induced effects usually were more clearly expressed after extended (20 h) than after acute (5 h) culture periods. Addition of the hormones insulin or glucagon caused dose-dependent alterations in the flux of substrates to glucose and lactate. Rates of de novo synthesis of fatty acids from [14C]acetate were stimulated by insulin and inhibited by glucagon during acute and extended incubation periods. Treatment of liver cells isolated from male trout for 72 h with estradiol induced vitellogenin production and secretion into the medium. However, the addition of insulin or dexamethasone drastically reduced this estrogen-induced vitellogenesis. These results indicate that trout liver cells cultured in defined medium maintain central metabolic pathways, including glycolysis, gluconeogenesis, lipogenesis, and vitellogenesis as well as their responsiveness to various hormones, for at least 72 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Clastogenic effects of the dithiocarbamate fungicides thiram and ziram in Chinese hamster cell lines cultured in vitro

We report here the results obtained using the dithiocarbamate fungicides thiram and ziram to investigate the induction of chromosomal aberrations (CAs) in Chinese hamster ovary (CHO) cells both in the absence and presence of S9 metabolism, and in a Chinese hamster epithelial liver (CHEL) cells which retain metabolic competence to activate different classes of promutagens/procarcinogens. Both thiram and ziram proved to be strong chromosome breaking agents in the CHEL cells and CHO cells in the presence of S9 metabolism. These findings suggest that thiram and ziram require metabolic conversion to become genetically active, and corroborate the evidence that CHEL cells are suitable to activate and detect a broad spectrum of chemical procarcinogens including these two pesticides.

Metabolic assessment of a neuron‐enriched fraction of rat cerebrum using high‐resolution1H and13C NMR spectroscopy

This study explored the utility of 1H and 13C magnetic resonance spectroscopy to study a neuron-enriched preparation made from rat cerebrum. The preparation contained high concentrations of N-acetylaspartate and gamma-aminobutyric acid and low concentrations of glutamine, indicating that it was in fact rich in neuronal cytosol. This was confirmed by immunohistochemical studies with antibodies to neuronal and glial markers. A method of metabolite quantification based on the creatine signal yielded metabolite concentrations similar to those of rat cerebrum, whereas concentrations based on the metabolite/protein ratio were five times lower, suggesting that much protein in the preparation was not associated with functioning cytoplasm. The metabolic competence of the preparation was assessed by quantitative measurements of its ability to convert 1-13C-glucose into lactate, glutamate, aspartate, and other metabolites under well oxygenated conditions for 30 min. Calculated from the creatine standard, the mean glycolytic rate was the same as in a synaptosomal preparation studied under similar conditions and the same as rat cerebrum in vivo. Tricarboxylic acid cycle flux occurred at half the rate observed in the synaptosomal preparation and 16% of the basal cerebral metabolic rate in vivo.

V79 Chinese hamster cells genetically engineered for cytochrome P450 and their use in mutagenicity and metabolism studies

V79 Chinese hamster cells are being genetically engineered for stable expression of cytochromes P450 using a SV40 early promoter containing plasmid as the eukaryotic expression vector. V79 cells lack endogenous cytochrome P450 activity. Therefore, genetically engineered V79 cell lines are defined for the cytochrome P450 isoform acquired upon cDNA mediated gene transfer. V79 cells have a longstanding tradition as indicator cells for various biological endpoints, e.g. mutation, chromosomal aberration, cytotoxicity. The genetically engineered V79 cells combine these useful biological endpoints with metabolic competence. In this sense, V79 cell lines genetically engineered for stable expression of cytochromes P450 present newly developed tools for studying and understanding metabolism related problems in toxicology and pharmacology.

Assessment of the Functional Integrity of Hepatocytes: A Brief Review

A variety of approaches to assessment of cellular integrity exist, based on tests of integrity of the plasma membrane, tests of metabolic competence, and asessment of morphology. By definition, such approaches address different aspects of cellular integrity and hence are not interchangeable indices of cellular integrity. Accordingly, it would be most appropriate to characterise hepatocyte preparations on the basis of more than just trypan blue dye exclusion (a test of plasma membrane integrity) as is customary. A scheme for the choice of the most appropriate mix of tests of cellular integrity is presented.

Preservation of the rate and profile of xenobiotic metabolism in rat hepatocytes stored in liquid nitrogen

A simple procedure for cryopreservation of rat hepatocytes that allows recovery of viable cells retaining activities of phase I and phase II drug metabolism equivalent to freshly isolated cells is described. The cooling process was initiated 30 min after incubation of freshly isolated hepatocytes at 37° in Krebs-Ringer bicarbonate buffer containing 15 mM glucose to allow for metabolic equilibration. At the end of this period, hepatocyte suspensions were supplemented with 1.7% albumin, 13.3% dimethyl sulfoxide, and the synthetic buffers, 3-[-morpholino]propanesulfonic acid (MOPS) and N-[2-hydroxyethyl]piperazine- N′-[2-ethanesulfon acid] (HEPES). Hepatocytes were cooled in a step-wise manner to −196° by holding the cells for 1 hr at −20° and then for 1 hr at −70° before transfer into liquid nitrogen. After thawing and removal of damaged cells by centrifugation in Percoll, the total recovery of viable hepatocytes subjected to freezing was about 42%. The contents of ATP, ADP, and AMP were not altered significantly in cells stored in liquid nitrogen. The metabolic competence of cryopreserved hepatocytes was further confirmed by their ability to synthesize urea from NH 4Cl and ornithine at the same high rate that was observed in freshly isolated cells (693 ± 68 and 740 ± 68 nmol · mg dry wt −1·hr −1, respectively). Similarly, cryopreservation did not affect drug-metabolizing systems as indicated by the metabolism of benzo[ a]pyrene and 7-ethoxycoumarin, two model substrates. In both freshly isolated and cryopreserved hepatocytes, 7-ethoxycoumarin was O-deethylated to 7-hydroxycoumarin at essentially the same rates (8.66 ± 0.75 and 8.25 ± 0.53 nmol · mg dry wt −1·hr −1, respectively) and 7-hydroxycoumarin accumulated in hepatocyte suspensions almost exclusively in the conjugated form. The storage of hepatocytes in liquid nitrogen also did not affect the complex metabolism of benzo[ a]pyrene to total oxygenated metabolites and, more importantly, to metabolites conjugated with glutathione, glucuronic acid, and sulfuric acid. Thus, cryopreserved hepatocytes represent a valid and convenient model to study drug biotransformation in intact cells.

Alteratioon in proliferative and endocrine responsiveness of human mammary carcinoma cells by prototypic tumor-suppressing agents

The experiments performed in this study were designed to establish that (1) acquisition of anchorage-independent growth, a biological characteristic of tumorigenically transformed phenotype, can be modulated by prototypic tumor-suppressing agents, and (2) modulation of growth is influenced by the metabolic competence of the cells to biotransform estradiol. MCF-7 human breast carcinoma cells exhibited linear cell proliferative kinetics with a 41-hour population doubling time, and a 15% colony-forming efficiency in 0.33% agar. Indole-3-carbinol (I3C), a naturally occurring tumor-suppressive agent; tamoxifen (TAM), an antiestrogenic agent; and 4-hydroxytamoxifen (4-OHTAM), a metabolite of TAM, demonstrated 73.7%, 72.5%, and 89.9% suppression in anchorage-independent growth of MCF-7 cells, respectively. At the metabolic level, 13C and 4-OHTAM induced 2.3-fold ( P < 0.001) and 1.3-fold increase ( P = 0.001) relative to their own controls in the extent of 2-hydroxylation of estradiol. The results indicate that growth inhibition by I3C, TAM, and 4-OMTAM may in part be due to altered estradiol metabolism in MCF-7 cells. Thus, anchorage-independent growth and altered biotransformation of estradiol may constitute useful cellular and endocrine markers to evaluate the biological response of chemosuppressive agents.

Cytogenetic effects of promutagens in genetically engineered V79 chinese hamster cells expressing cytochromes P450

V79 Chinese hamster cell lines genetically engineered to express rat CYP2B1, CYP1A1, CYP1A2, and their parental cell lines V79-MZ, without acetyltransferase, and V79-NH, with acetyltransferase, were studied for chromosome aberrations and sister chromatid exchange induced by aflatoxin B 1, cyclophosphamide, benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene and dimethylnitrosamine. The parental V79 cell lines did not show clastogenic effects. Significant clastogenic effects were observed after an 18 h exposure to aflatoxin B 1 and cyclophosphamide in CYP2B1 expressing cells, to benzo[a]pyrene in CYP1A1 and CYP1A2 expressing cells, to 7,12-dimethylbenz[a]anthracene and dimethylnitrosamine in cells, expressing CYP1A2 with or wothout acetyltransferase, and to cyclophosphamide in cells expressing both CYP1A2 and acetyltransferase. A significant sister chromatid exchange inducing effect was found after a 24 h exposure in each of the genetically engineered cell lines, except for benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in CYP2B1 expressing cells, and for benzo[a]pyrene in cells expressing both CYP1A2 and acetyltransferase. Thus, a battery of cell lines genetically engineered for metabolic competence may serve as a tool for investigating chromosomal changes induced by activated xenobiotics.

Cytotoxicity of tacrine and velnacrine metabolites in cultured rat, dog and human hepatocytes.

Clinical trials with tacrine (THA) and its principal (1-OH) metabolite (velnacrine) for the treatment of Alzheimer's disease have been hampered by adverse hepatic events that were undetected in preclinical studies. As part of integrated in vivo/in vitro efforts to characterize the role of metabolites in these events, cultured cells were evaluated for their suitability for further mechanistic studies. The relative cytotoxic potentials of THA, three monohydroxy metabolites of THA (including velnacrine, a racemate), the two velnacrine enantiomers, and several known and suspected dihydroxy velnacrine metabolites were determined. Cytotoxicity was evaluated in 24-hour cultures by morphology and by the Neutral Red Uptake Assay. All test articles were evaluated in primary rat hepatocytes and in a human hepatoma cell line (HepG2). THA and velnacrine were also tested in a rat hepatoma cell line (H4) and in primary dog hepatocytes. The metabolic competency of each cell type was determined. Sensitivity to THA and velnacrine was greatest in H4 cells, followed by primary rat and HepG2 cells; dog cells were least sensitive. In HepG2 cells, THA was clearly more cytotoxic (LC50:54 micrograms/ml) than its monohydroxy metabolites (LC50 values: 84 to 190 micrograms/ml); dihydroxy velnacrine metabolites were the least cytotoxic (LC50 values:251 to 434 micrograms/ml); the relative order was comparable in primary rat hepatocytes. Roles for reactive metabolites and/or altered metabolic capabilities of Alzheimer's patients are suggested.

Morphology and monoterpene biosynthetic capabilities of secretory cell clusters isolated from glandular trichomes of peppermint (Mentha piperita L.)

Secretory cells were isolated from the monoterpene-producing glandular trichomes (peltate form) of peppermint as clusters of eight cells each. These isolated structures were shown to be non-specifically permeable to low-molecular-weight, water-soluble cofactors and substrates. Short incubation periods with the polar dye Lucifer yellow iodoacetamide (Mr=660) resulted in a uniform staining of the cytoplasm, with exclusion of the dye from the vacuole. The molecular-weight exclusion limit for this permeability was shown to be less than approx. 1800, based on exclusion of fluorescein-conjugated dextran (Mr ∼ 1800). Intact secretory cell clusters very efficiently incorporated [(3)H]geranyl pyrophosphate into monoterpenes. The addition of exogenous cofactors and redox substrates affected the distribution of monoterpenes synthesized from [(3)H]geranyl pyrophosphate, demonstrating that the cell clusters were permeable to these compounds and that the levels of endogenous cofactors and redox substrates were depleted in the isolated cells. When provided with the appropriate cofactors, such as NADPH, NAD(+), ATP, ADP and coenzyme A, the isolated secretory cell clusters incorporated [(14)C]sucrose into monoterpenes, indicating that these structures are capable of the de-novo biosynthesis of monoterpenes from a primary carbon source, and that they maintain a high degree of metabolic competence in spite of their permeable nature.