Search

Reactive oxygen species (ROS) and oxidative damage to biomolecules have been postulated to be involved in the causation and progression of several chronic diseases, including cancer and cardiovascular diseases, the two major causes of morbidity and mortality in Western world. Consequently dietary antioxidants, which inactivate ROS and provide protection from oxidative damage are being considered as important preventive strategic molecules. Carotenoids have been implicated as important dietary nutrients having antioxidant potential, being involved in the scavenging of two of the ROS, singlet molecular oxygen (1O2) and peroxyl radicals generated in the process of lipid peroxidation. Carotenoids are lipophilic molecules which tend to accumulate in lipophilic compartments like membranes or lipoproteins. Chronic ethanol consumption significantly increases hydrogen peroxide and decreases mitochondrial glutathione (GSH) in cells overexpressing CYP2E1. The depletion of mitochondrial GSH and the rise of hydrogen peroxide are responsible for the ethanol-induced apoptosis. Increased intake of lycopene, a major carotenoid in tomatoes, consumed as the all-trans-isomer attenuates alcohol induced apoptosis in 2E1 cells and reduces risk of prostate, lung and digestive cancers. Cancer-preventive activities of carotenoids have been associated as well as with their antioxidant properties and the induction and stimulation of intercellular communication via gap junctions which play a role in the regulation of cell growth, differentiation and apoptosis. Gap junctional communication between cells which may be a basis for protection against cancer development is independent of the antioxidant property.

Copper is a trace element, important for the function of many cellular enzymes. Copper ions can adopt distinct redox states oxidized Cu(II) or reduced (I), allowing the metal to play a pivotal role in cell physiology as a catalytic cofactor in the redox chemistry of enzymes, mitochondrial respiration, iron absorption, free radical scavenging and elastin cross-linking. If present in excess, free copper ions can cause damage to cellular components and a delicate balance between the uptake and efflux of copper ions determines the amount of cellular copper. In biological systems, copper homeostasis has been characterized at the molecular level. It is coordinated by several proteins such as glutathione, metallothionein, Cu-transporting P-type ATPases, Menkes and Wilson proteins and by cytoplasmic transport proteins called copper chaperones to ensure that it is delivered to specific subcellular compartments and thereby to copper-requiring proteins.

Reduced glutathione (GSH) is the most prevalent non-protein thiol in animal cells. Its de novo and salvage synthesis serves to maintain a reduced cellular environment and the tripeptide is a co-factor for many cytoplasmic enzymes and may also act as an important post-translational modification in a number of cellular proteins. The cysteine thiol acts as a nucleophile in reactions with both exogenous and endogenous electrophilic species. As a consequence, reactive oxygen species (ROS) are frequently targeted by GSH in both spontaneous and catalytic reactions. Since ROS have defined roles in cell signaling events as well as in human disease pathologies, an imbalance in expression of GSH and associated enzymes has been implicated in a variety of circumstances. Cause and effect links between GSH metabolism and diseases such as cancer, neurodegenerative diseases, cystic fibrosis (CF), HIV, and aging have been shown. Polymorphic expression of enzymes involved in GSH homeostasis influences susceptibility and progression of these conditions. This review provides an overview of the biological importance of GSH at the level of the cell and organism.

Polyphenols are the most abundant antioxidants in our diets. The main classes of polyphenols are phenolic acids (mainly caffeic acid) and flavonoids (the most abundant in the diet are flavanols (catechins plus proanthocyanidins), anthocyanins and their oxidation products), which account for one- and two-thirds, respectively. Polyphenols are reducing agents, and together with other dietary reducing agents, such as vitamin C, vitamin E and carotenoids, referred to as antioxidants, protect the body's tissues against oxidative stress and associated pathologies such as cancers, coronary heart disease and inflammation. The biological properties, bioavailability, antioxidant activity, specific interactions with cell receptors and enzymes, are related to the chemical structure of polyphenols. It is, therefore, essential to know the nature of the main polyphenols ingested, their dietary origin, the amounts consumed in different diets, their bioavailability and the factors controlling their bioavailability.

Folate (folic acid, folacin) is an essential vitamin that is found in nature. Folates contain the core chemical structure of pteroylglutamic acid, but vary in their state of reduction, the single carbon moiety they bear and/or the length of the glutamate chain. At least 50% of whole body folate is stored in the liver. The influence of intracellular folate concentration depends largely on dietary intake. The supply of folate depends primarily on the quantity and bioavailability of ingested folate and the rate of loss by urinary and fecal routes and through catabolism.

Summary Audiogenic seizures associated with loss of weight, prostration, piloerection, palpebral ptosis and motor deficiency were induced after sound stimulation of determined frequency and amplitude in magnesium-deficient DBA/2 mice. These symptoms were maintained when standard diet conditions (1700 ppm Mg2+) were restored. In contrast, mice were protected from audiogenic seizure in a dose related manner when Crassostrea gigas extract (JCOE) were added to the diet for 10 consecutive days. Although a rational explanation for this protective effect has not yet been determined, it is assumed that it might be due to a chelating complex formed between Mg2+ and taurine, which enhance the uptake of Mg2+. © 1998 Elsevier, Paris.

The amino-acid composition of Crassostera gigas extract powder (JCOE, Japan clinic oyster extract) is high in glutamic acid and taurine. In order to study whether these components present in JCOE could be contributory factors in enhancing cellular glutathione-stimulating hormone (GSH) and glutathione S-transferase (GST), HL60 cells were exposed to increasing concentrations of JCOE. Statistically significant increases in GSH occurred at extract concentrations of 0.05 and 0.1% with no significant change in GST activity. Because of the high taurine levels measured in the extract, the effects of micromolar taurine supplementation of the medium were studied. A modest, but not statistically significant increase in both GSH levels and GST activity was measured. We therefore conclude that while the taurine present in JCOE could be a contributory factor in enhancing GSH and GST, it was not the only stimulus.

In continuous Friend leukemia cell exposure to tTAuP, the IC50 was 0.2 μM whereas in cells exposed 15 or 60 min to tTAuP followed by 72 h in drug-free medium the IC50 was 2.2 and 1.3 μM respectively. A combination of tTAuP and cisplatin (CDDP) is shown to be more active than either agent alone. Intracellular accumulation studies analysed by SXRF have shown that to achieve a cytotoxic effect, large concentrations of gold are necessary to accumulate in both the nuclear and cytoplasmic fraction. However, at cytostatic doses (1 μM), tTAuP has no effect on the cell cycle but does affect DNA synthesis. At a higher dose, greater than that necessary to induce cytotoxicity, it produces DNA damage, as observed by alkaline elution method. When cells were exposed to toxic doses of tTAuP (10 μM) in the presence of albumin, cytotoxicity was significantly reduced. Similar results were obained when cells were co-treated with L-cysteine, dithiothreitol or reduced gluthatione. Reduced cytotoxic effect can be related to the interaction with free thiol groups. According to these data it is concluded that tTAuP is highly effective in vitro; whether it is active in vivo remains to be determined.

In adriamycin-resistant murine (FLC) and human (K562) leukemia cells, phosphatidyicholine increases and phosphatidylethanolamine decreases compared to adriamycin-sensitive lines. This change is due to an increase in phosphatidylethanolamine methylation. The choline pathway of phosphatidyicholine biosynthesis is also disturbed in resistant cells with a blocking step of CDP-phosphocholine transferase and a decrease in sphingomyelinase activity. These changes in phospholipid metabolism are suggested to be responsible for the changes in membrane fluidity reported previously (Tapiero et al , 1986) for resistant cells. phosphatidyicholine / sphingomyelin / MDR + leukemia cells

The analytical conditions in PIXE and SXRF are studied in order to optimize the detection of platinum and gold compounds in the cell and in their subcellular fractions. Calibration curves, reproducibility and detection limits were obtained with both techniques and compared. The best detection limit was achieved with SXRF. Preliminary data on the cellular pharmacokinetics of cisplatinum and gold selective distribution in nuclei were obtained by the above techniques.

A strong association between elevated plasma low-density-lipoprotein (LDL) and the development of cardiovascular diseases (CVD) has been established. Oxidation of LDL (Ox-LDL) promotes vascular dysfunction, enhances the production and release of inflammatory mediators such as reactive oxygen species and contribute to the initiation and progression of atherosclerosis. In addition, Ox-LDL enhances the production and release of tumor necrosis factor (TNF-α), interleukin (IL)-6, arachidonic acid metabolites and nitric oxide (NO) that are responsible for various human pathologies including cancer. Organosulfur compounds (OSC) from alliaceae modulate the glutathione (GSH) redox cycle and inhibits NFκ-B activation in human T cells. Furthermore, OSC bioactivities include antioxidant, antibacterial, anticarcinogenic, antiatherogenic, immunostimulatory, and liver protection potential.

Sulfur containing amino acids contribute substantially to the maintenance and integrity of cellular systems by influencing cellular redox state and cellular capacity to detoxify toxic compounds, free radicals and reactive oxygen species. Methionine and cysteine are the two primary sulfur-containing amino acids in mammals. Methionine is an essential amino acid, obtained by dietary intake while cysteine is non-essential and a metabolite of methionine metabolism. Each of these amino acids contributes significantly to the cellular pool of organic sulfur and generally to sulfur homeostasis as well as playing a significant role in regulation of one carbon metabolism. Genetic defects in the enzymes regulating sulfur pools produce a variety of human pathologies, including homo- and cystinuria, homo- and cysteinemia, and neural tube defects. In addition, thiol imbalance has been associated with multiple disorders, including vascular disease, Alzheimer’s, HIV and cancer. Possible treatments to restore the thiol balance are also discussed.

Zinc is one of the most abundant nutritionally essential elements in the human body. It is found in all body tissues with 85% of the whole body zinc in muscle and bone, 11% in the skin and the liver and the remaining in all the other tissues. In multicellular organisms, virtually all zinc is intracellular, 30-40% is located in the nucleus, 50% in the cytoplasm, organelles and specialized vesicles (for digestive enzymes or hormone storage) and the remainder in the cell membrane. Zinc intake ranges from 107 to 231 micromol/d depending on the source, and human zinc requirement is estimated at 15 mg/d. Zinc has been shown to be essential to the structure and function of a large number of macromolecules and for over 300 enzymic reactions. It has both catalytic and structural roles in enzymes, while in zinc finger motifs, it provides a scaffold that organizes protein sub-domains for the interaction with either DNA or other proteins. It is critical for the function of a number of metalloproteins, inducing members of oxido-reductase, hydrolase ligase, lyase family and has co-activating functions with copper in superoxide dismutase or phospholipase C. The zinc ion (Zn(++)) does not participate in redox reactions, which makes it a stable ion in a biological medium whose potential is in constant flux. Zinc ions are hydrophilic and do not cross cell membranes by passive diffusion. In general, transport has been described as having both saturable and non-saturable components, depending on the Zn(II) concentrations involved. Zinc ions exist primarily in the form of complexes with proteins and nucleic acids and participate in all aspects of intermediary metabolism, transmission and regulation of the expression of genetic information, storage, synthesis and action of peptide hormones and structural maintenance of chromatin and biomembranes.

Coronary heart disease is a major health problem in developed countries. Many studies have shown that elevated serum concentrations of total or low-density-lipoprotein cholesterol (LDL cholesterol) are high risk factors, whereas high concentrations of high-density-lipoprotein cholesterol (HDL cholesterol) or a low LDL to HDL cholesterol ratio may protect against coronary heart disease. Plant sterols and stanols derived from vegetable oils or wood pulp have been shown to lower total and LDL cholesterol levels in humans by inhibiting cholesterol absorption from the intestine. These findings may lead to new therapeutic options to treat hypercholesterolemia. In addition, phytosterols may influence cell growth and apoptosis of tumor cells. However, they can interfere with the absorption of fat soluble vitamins and carotenoids.

Anthracyclines are the most commonly used classes of anticancer agents in chemotherapy. Development of resistance to these molecules is one of the major reasons for treatment failure. The overexpression of the membrane transporter P-glycoprotein (P-gp) is among the principal mechanisms involved in this phenomenon. This pump, which is responsible for the multidrug resistance (MDR) phenotype, decreases the toxicity of a wide range of unrelated anticancer drugs by increasing their cellular efflux. Structure-activity relationship experiments have shown that the positively charged amino group of the anthracyclines could be responsible for their transport by P-gp. Here, we used three new anthracyclines that shared the same chromophore but differed by the degree of N -methylation of their sugar moiety. Oxaunomycin (OXN) possessed a non-methylated amino group, while LB-1 was monomethylated and β-clamycin T (BCT) was dimethylated. In sensitive cells (FLC), reduced cytotoxicity was related to the level of N -methylation; whereas in resistant cells (DOX-RFLC 1 and DOX-RFLC 2 ) overexpressing different levels of P-gp, increased N -methylation enhanced anthracycline cytotoxicity. Decreased resistance in DOX-RFLCs was associated with an increased drug accumulation due to a reduced cellular efflux. As expected, the MDR modulator verapamil decreased resistance to these anthracyclines by increasing the cellular accumulation. These results suggest that N -methylation of anthracyclines circumvents resistance by diminishing drug transport by P-gp in MDR-positive cells. These observations could be the consequence of the steric hindrance created by the methyl group(s) which may impair the interaction between the positively charged amino group and the active site of P-gp.

Selenium (Se) is an essential trace element for animals and humans that is obtained from dietary sources including cereals, grains and vegetables. The Se content of plants varies considerably according to its concentration in soil. Plants convert Se mainly into Se-methionine (Se-Met) and incorporate it into protein in place of methionine (Met). Selenocystine (Se-Cys), methyl-Se-Cys and gamma-glutamyl-Se-methyl-Cys are not significantly incorporated into plant protein and are at relatively low levels irrespective of soil Se content. Higher animals are unable to synthesize Se-Met and only Se-Cys was detected in rats supplemented with Se as selenite. Renal regulation is the mode by which whole body Se is controlled. Se is concentrated in hair and nail and it occurs almost exclusively in organic compounds. The potentiating effect of Se deficiency on lipid peroxidation is enhanced in some tissues by concurrent deficiency of copper or manganese. In the in vitro system, the chemical form of Se is an important factor in eliciting cellular responses. Although the cytotoxic mechanisms of selenite and other redoxing Se compounds are still unclear, it has been suggested that they derive from their ability to catalyze the oxidation of thiols and to produce superoxide simultaneously. Selenite-induced cytotoxicity and apoptosis in human carcinoma cells can be inhibited with copper (CuSO(4)) as an antioxidant. High doses of selenite result in induction of 8-hydroxydeoxyguanosine (8-OHdG) in mouse skin cell DNA and in primary human keratinocytes. It may cause DNA fragmentation and decreased DNA synthesis, cell growth inhibition, DNA synthesis, blockade of the cell cycle at the S/G(2)-M phase and cell death by necrosis. In contrast, in cells treated with methylselenocyanate or Se methylselenocysteine, the cell cycle progression was blocked at the G(1) phase and cell death was predominantly induced by apoptosis.

L-Arginine (Arg) is classified as an essential amino acid for birds, carnivores and young mammals and a conditionally essential amino acid for adults. It is converted by arginase to L-ornithine, a precursor of polyamines and urea, which is important in the urea cycle. Arg serves as a precursor for creatine, which plays an essential role in the energy metabolism of muscle, nerve and testis and accounts for Arg catabolism and for the synthesis of agmatine and proteins. Via its ability to increase growth hormone secretion it influences immune function. Depending on nutritional status and developmental stage, normal plasma Arg concentrations in humans and animals range from 95 to 250 micromol/l. Systemic or oral Arg administration has been shown to improve cardiovascular function and reduce myocardial ischemia in coronary artery disease patients. It reduces blood pressure and renal vascular resistance in essential hypertensive patients with normal or insufficient renal function. Although Arg plasma concentrations are not altered in hypercholesterolemic individuals, oral or intravenous Arg administration can reverse endothelial dysfunction in hypercholesterolemic patients and in cigarette smokers. The main importance of Arg is attributed to its role as a precursor for the synthesis of nitric oxide (NO), a free radical molecule that is synthesized in all mammalian cells from L-Arg by NO synthase (NOS). NO appears to be a major form of the endothelium-derived relaxing factor (EDRF). NO and EDRF share similar chemical and pharmacological properties and are derived from the oxidation of a terminal guanidine group of L-Arg. Various mechanisms have been implicated in the defect in vascular relaxation. These include, increased diffusional barrier for NO, L-Arg depletion, altered levels of reactive oxygen, inactivation of NO by superoxide anions (O2-). The independent reactions of O2-, NO and their reaction yielding peroxynitrite are critical in the initiation and maintenance of the atherosclerotic state and contribute to the defect in vasorelaxation. NO also plays a role as a neurotransmitter, mediator of immune response and as signaling molecule. The NO synthesized by iNOS in macrophages contributes to their cytotoxic activity against tumor cells, bacteria and protozoa. Our aim here is to review on some amino acids with high functional priority such as Arg and to define their effective activity in human health and pathologies.

Glutamine and glutamate with proline, histidine, arginine and ornithine, comprise 25% of the dietary amino acid intake and constitute the "glutamate family" of amino acids, which are disposed of through conversion to glutamate. Although glutamine has been classified as a nonessential amino acid, in major trauma, major surgery, sepsis, bone marrow transplantation, intense chemotherapy and radiotherapy, when its consumption exceeds its synthesis, it becomes a conditionally essential amino acid. In mammals the physiological levels of glutamine is 650 micromol/l and it is one of the most important substrate for ammoniagenesis in the gut and in the kidney due to its important role in the regulation of acid-base homeostasis. In cells, glutamine is a key link between carbon metabolism of carbohydrates and proteins and plays an important role in the growth of fibroblasts, lymphocytes and enterocytes. It improves nitrogen balance and preserves the concentration of glutamine in skeletal muscle. Deamidation of glutamine via glutaminase produces glutamate a precursor of gamma-amino butyric acid, a neurotransmission inhibitor. L-Glutamic acid is a ubiquitous amino acid present in many foods either in free form or in peptides and proteins. Animal protein may contain from 11 to 22% and plants protein as much as 40% glutamate by weight. The sodium salt of glutamic acid is added to several foods to enhance flavor. L-Glutamate is the most abundant free amino acid in brain and it is the major excitatory neurotransmitter of the vertebrate central nervous system. Most free L-glutamic acid in brain is derived from local synthesis from L-glutamine and Kreb's cycle intermediates. It clearly plays an important role in neuronal differentiation, migration and survival in the developing brain via facilitated Ca++ transport. Glutamate also plays a critical role in synaptic maintenance and plasticity. It contributes to learning and memory through use-dependent changes in synaptic efficacy and plays a role in the formation and function of the cytoskeleton. Glutamine via glutamate is converted to alpha-ketoglutarate, an integral component of the citric acid cycle. It is a component of the antioxidant glutathione and of the polyglutamated folic acid. The cyclization of glutamate produces proline, an amino acid important for synthesis of collagen and connective tissue. Our aim here is to review on some amino acids with high functional priority such as glutamine and to define their effective activity in human health and pathologies.

Linoleic and alpha-linolenic acids, obtained from plant material in the diet are the precursors in tissues of two families with opposing effects which are referred to as “essential fatty acids” (EFA): arachidonic acid (AA) and pentaene (eicosapentaenoic acid: EPA) and hexaene (docosahexaenoic acid: DHA) acids. The role of EFA is crucial, without a source of AA or compounds which can be converted into AA, synthesis of prostaglandins (PGs) by a cyclooxygenase (COX) enzyme would be compromised, and this would seriously affect many normal metabolic processes. COX, also known as prostaglandin endoperoxide synthase (Pghs) or as prostaglandin G/H synthase, is a key membrane bound enzyme responsible for the oxidation of AA to PGs. Two COX isoforms have been identified, COX-1 and COX-2 that form PGH 2 , a common precursor for the biosynthesis of thromboxane A 2 (TxA 2 ), prostacyclin (PGI 2 ) and PGs (PGD 2 , PGE 2 , PGF 2α . COX-1 enzyme is expressed constitutively in most cells and tissues. Its expression remains constant under either physiological or pathological conditions controlling synthesis of those PGs primarily involved in the regulation of homeostatic functions. In contrast, COX-2 is an intermediate response gene that encodes a 71-kDa protein. COX-2 is normally absent from most cells but highly inducible in certain cells in response to inflammatory stimuli resulting in enhanced PG release. PGs formed by COX-2 primarily mediate pain and inflammation but have multiple effects that can favour tumorigenesis. They are more abundant in cancers than in normal tissues from which the cancers arise. COX-2 is a participant in the pathway of colon carcinogenesis, especially when mutation of the APC (Adenomatous Polyposis Coli) tumour suppressor gene is the initiating event. In addition, COX-2 up-regulation and elevated PGE 2 levels are involved in breast carcinogenesis. It seems that there is a correlation between COX-2 level of expression and the size of the tumours and their propensity to invade underlying tissue. Inhibition by non-steroidal anti-inflammatory drugs (NSAIDs) of COX enzymes which significantly suppress PGE 2 levels, reduced breast cancer incidence and protected against colorectal cancer. Therefore it is suggested that consumption of a diet enriched in n-3 PUFA (specifically EPA and DHA) and inhibition of COX-2 by NSAIDs may confer cardioprotective effects and provide a significant mechanism for the prevention and treatment of human cancers.

A report from the World Health Organization estimates that 46% of the world's 5- to 14-year-old children are anemic. In addition, 48% of the world's pregnant women are anemic. A majority of these cases of anemia are due to iron deficiency. Our aim here is to review the latest data on iron regulatory mechanisms, iron sources and requirements. Human and animal studies have shown that amino acids and peptides influence iron absorption from the intestinal lumen. Inter-organ transport and uptake of nonheme iron is largely performed by the complex transferring-transferring receptor system. Moreover, the discovery of cytoplasmic iron regulatory proteins (IRPs) has provided a molecular framework from which we understand the coordination of cellular iron homeostasis in mammals. IRPs and the iron responsive elements (IREs) to which they bind allow mammals to make use of the essential properties of iron while reducing its potentially toxic effect. Physiologic iron requirements are three times higher in pregnancy than they are in menstruating women (approximately 1200 mg must be acquired from the body's iron store or from the diet by the end of pregnancy). The administration of iron supplements weekly instead of daily in humans has been proposed and is being actively investigated as a viable means of controlling iron deficiency in populations, including pregnant women.

Exposure to oxidant molecules issued from the environment (pollution, radiation), nutrition, or pathologies can generate reactive oxygen species (ROS for example, H2O2, O2-, OH). These free radicals can alter DNA, proteins and/or membrane phospholipids. Depletion of intracellular antioxidants in acute oxidative stress or in various diseases increases intracellular ROS accumulation. This in turn is responsible for several chronic pathologies including cancer, neurodegenerative or cardiovascular pathologies. Thus, to prevent against cellular damages associated with oxidative stress it is important to balance the ratio of antioxidants to oxidants by supplementation or by cell induction of antioxidants.

Summary Previously we have detected a stimulatory effect on immunoglobulin (IgG) synthesis when hybridoma cells were treated with doxorubicin. In order to determine whether this is a general property of anthracycline, we have selected three analogs doxorubicin (DOX), pirarubicin (THP-DOX) and aclarubicin (ACR) — which differ mainly in the methylation state of their amino sugars. Cell cycle analysis by flow cytometry and drug localization by scanning confocal microscopy were also performed. The results show that when cells (UN2 hybridoma B cells) were exposed to subtoxic doses of DOX or THP (with unmethylated amino sugars), a strong increases in IgG secretion, heavy (H) and light (L) chain synthesis and the corresponding mRNA levels were induced. Furthermore these two drugs arrested the cells in the G2/M phase of the cell cycle. In contrast, exposure to ACR (with its methylated amino sugar) at similar subtoxic doses induced a blockade of cells in the Gl phase with no increase of IgG synthesis. at the subtoxic doses used, all three drugs could still be detected in the nucleus as well as in the cytoplasm, as determined by confocal laser microscopy. Thus, the relationship between cell cycle blockade, IgG stimulation and anthracycline structure is suggested by these results. © 1998 Elsevier, Paris.

Since several in vitro and animal studies of an extract from Crassostrea gigas (JCOE) have demonstrated its antioxidant properties and other interesting effects, a preliminary human trial was carried out. Seven healthy male volunteers aged 23-37 received orally 3 x 2 capsules of JCOE per day for 8 days. On days 0, 1, 4, 8 and 15 (7 days after completion of the schedule) blood samples were drawn and the antioxidant potential of serum was tested. A statistically significant increase in the buffering effects of serum against hemoglobin (Hb) and lactate dehydrogenase (LDH) release from red blood cells treated with the free radical generator azobis amidino propane (AAPH) was found following JCOE treatment. At 8 days, the oxidative effects were reduced by90% of the pretreatment values. In these same individuals, serum levels of reduced glutathione were increased by an average of 1.5-fold over the time course of treatment. It is concluded that in normal human volunteers, JCOE capsules provide an orally available formula for enhancing the antioxidant capacity of blood serum. While the extract is known to contain some direct acting antioxidant components, at least a portion of the protective effect is facilitated by enhancement of GSH biosynthesis.

Male Sprague Dawley rats received various amounts of extract of Crassostrea gigas by gavage every day for 2 weeks or one month. At these times, groups of animals were sacrificed and samples of major organs analyzed for levels of glutathione (GSH) and glutathione S-transferase (GST) activities. Following the two week protocol, GSH levels were significantly increased in the mucosa of the large intestine; at one month the small intestine and spleen were elevated. GST activity increased in liver under both schedules and at one month, activity was also elevated in kidney and small intestine. Since the Crassostrea gigas extract contains high levels of a variety of important amino acids, it is concluded that biologically available peptides are taken up in target organs and stimulate GSH metabolism. Enhanced levels of GSH and associated enzymes may contribute to a more effective detoxification phenotype, thus providing enhanced chemoprotective capacity.

Structure/functional studies previously reported showed that in a series of simple organic cations in which the charge is delocalized, an aromatic ring and a minimal degree of lipophilicity (log P > −1) were required for recognition by murine cells which express P-glycoprotein (p-gp)-mediated multidrug resistance (MDR). In the present report we find that 3H-octylpyridinium, the simple aromatic cation which has been shown to be preferentially toxic to MDR− as compared to MDR+ cells, accumulates 4.7-fold greater in the MDR− cell line. In contrast, we find that 3H-guanidinium which displays no selective toxicity between MDR+ and MDR− cells, shows no significant uptake differences between these two cell types. We also present data which demonstrate that other organic cations which contain aromatic rings, a minimal degree of lipophilicity (log P> −1) and carry a delocalized (Rho 123) or shielded (triphenylmethyl phosphonium) positive charge, also accumulate to a greater degree in MDR− vs MDR+ cells. Additionally, we find that human cells which express p-gp MDR, have similar requirements for recognition of these simple compounds. In fact, the sensitivity profiles of these compounds closely correlate between murine and human cell lines. It was also found that none of the series of simple organic compounds tested showed modulatory activity in MDR+ cells, as assayed by monitoring retention of Rho 123. Thus, the requirements for MDR recognition vs those for MDR modulation are clearly distinguished with these simple structured compounds. In comparison, the calcium channel antagonist, verapamil, and a calcium channel agonist, Bay K 8644, both showed modulatory activity by increasing Rho 123 retention in MDR+ cells, further supporting the interpretation that verapamil’s modulation of MDR is unrelated to its action on calcium flux. Overall, the data presented here add further information for defining the structural requirements of compounds for their recognition by, or modulation of, human cells expressing p-gp-mediated MDR

Summary Peripheral blood mononuclear cells (PBMCs) are physiologically activated by interleukin (IL)-2. We found that oyster extract (JCOE) currently used as a functional nutrient enhanced in vitro the IL-2 dependent activation as measured by cell count, 3 H-thymidine uptake and up-regulation of a IL-2 receptor. In human immunodeficiency virus (HIV) seropositive individuals, this oyster extract-induced effect was marked in asymptomatic individuals with quasi-normal CD4 cell counts, but was weakly reflected in acquired immunodeficiency syndrome (AIDS) patients.

Positively charged doxorubicin (DOX) and non-positively charged anthracyclines, aclarubicin (ACR) and morpholino-carminomycin (KRN 8602), have been investigated with respect to pharmacological parameters, cytotoxicity, DNA damage and repair in DOX-sensitive and -resistant murine and human cells. Friend leukemia cells (FLC) resistant to high concentrations of doxorubicin (DOX-RFLC3) or daunorubicin (DNR-RFLC3) (1771 and 1543 fold resistance respectively) express less than 10 fold resistance to aclarubicin (ACR). In these cells, the intracellular accumulation of ACR is similar in sensitive and resistant cells. Resistance to ACR was not observed in either DOX-RFLC1 or DNR1 with a lower level of resistance (27 fold). Increased expression of a 170,000-dalton surface antigen (gp-170) was found to be correlated with the level of resistance. However, when the selective agent in ACR, despite the low level of resistance (2.8 fold) both high expression of gp 170 and resistance to DOX (77 fold) or DNR (62 fold) are observed. It is assumed therefore that induction of multidrug resistance phenotype can be achieved by compounds which do not display cross resistance with DOX or DNR. Reduced levels or absence of cross-resistance can be related to the electrical charge of the compound. This assumption is supported by further studies on DOX-sensitive or -resistant human K562 cells exposed to another non-positively charged anthracycline, KRN 8602. In the continuous presence of drug, K562/DOX were less resistant to KRN 8602 (2.9 fold) than to DOX (31 fold). After short time exposure followed by growth in drug-free medium, absence of cross-resistance to KRN 8602 has been observed in K562/DOX. Furthermore, accumulation experiments showed that high intracellular drug concentrations were rapidly achieved (within 15 min) in both DOX-sensitive and -resistant cells. In cells exposed to DOX, DNA single-strand break (DNA-SSBs) frequencies were related to time and drug concentration while those produced by KRN 8602 or ACR were maximal after short time incubation. DNA-SSBs produced by these anthracyclines are not repaired when cells are incubated in drug free medium. In DOX resistant cells, DNA-SSBs produced by DOX were repaired whereas those produced by ACR or KRN 8602 were not. It is suggested, therefore, that absence of cross resistance to various anthracyclines is related to differences in the chemical electrical charge, which may influence drug accumulation and DNA repair in resistant cells.

In continuous Friend leukemia cell exposure to tTAuP, the IC50 was 0.2 microM whereas in cells exposed 15 or 60 min to tTAuP followed by 72 h in drug-free medium the IC50 was 2.2 and 1.3 microM respectively. A combination of tTAuP and cisplatin (CDDP) is shown to be more active than either agent alone. Intracellular accumulation studies analysed by SXRF have shown that to achieve a cytotoxic effect, large concentrations of gold are necessary to accumulate in both the nuclear and cytoplasmic fraction. However, at cytostatic doses (1 microM), tTAuP has no effect on the cell cycle but does affect DNA synthesis. At a higher dose, greater than that necessary to induce cytotoxicity, it produces DNA damage, as observed by alkaline elution method. When cells were exposed to toxic doses of tTAuP (10 microM) in the presence of albumin, cytotoxicity was significantly reduced. Similar results were obtained when cells were co-treated with L-cysteine, dithiothreitol or reduced gluthatione. Reduced cytotoxic effect can be related to the interaction with free thiol groups. According to these data it is concluded that tTAuP is highly effective in vitro; whether it is active in vivo remains to be determined.

Structurally different biological and chemical agents have been shown to block the proliferation of human and mouse tumor cells and to induce the production of molecules that are usually synthesized by terminally-differentiated cells. For instance, anti-neoplastic drugs such as Ara-C or doxorubicin (DOX) promote the irreversible induction of hemoglobin (Hb) synthesis in human erythroleukemia K562 cells with a concomitant loss of proliferative capacity (Luisi-Deluca et al., 1984; Jeannesson et al., 1984; Mazouzi et al., 1991). Similarly, the treatment of mouse hybridoma B cells by low-doses of DOX induces the blockade of cell proliferation through an accumulation of the cells in the G2 + M phase of the cell cycle as well as a strong increase of the Immunoglobulin (Ig) production (Teillaud et al., 1989), suggesting a terminal differentiation in “plasma-like” cells.

To study cross-resistance to Photofrin (PF) photosensitization, a Friend leukaemia cell line (ADM-RFLC) with a high level of multi-drug resistance (MDR) and the parental sensitive cell line (FLC) have been used. PF uptake measured by HPLC shows a similar intracellular drug accumulation in both cell lines. The ID50s for cell growth inhibition by PF are also similar after exposure in the dark in the two cell lines, while after illumination they are slightly lower in ADM-RFLC than in FLC cells. Moreover, verapamil, known to reverse the MDR phenotype induced by P-glycoprotein over-expression (the drug efflux mechanism), affects equally ADM-RFLC and FLC cells sensitivity to PF. In addition, photodynamic treatment with PF did not reverse the resistance to rhodamine 123 and aclarubicin, but partly reverses resistance of ADM-RFLC cells to antitubulin drugs such as vinblastine or vincristine. These latter results could have clinical application in the treatment of tumours expressing the MDR phenotype.

We previously noted that a wide variety of drugs which are recognized by multidrug-resistant cells (MDR+) are positively charged. However, it remains unclear why and how such a large number of structurally different compounds can be distinguished by MDR+ cells. The majority of the diverse compounds subject to MDR are complex and thereby complicate definitive structure/function characterization of the P-glycoprotein-mediated MDR mechanism. Using a series of simple aromatic (alkypyridiniums) and nonaromatic (alkylguanidiniums) organic cations differing in their lipophilicity by stepwise additions of single alkyl carbons, we demonstrate by growth inhibition studies that a single aromatic moiety and a critical degree of lipophilicity (log P-1) are required for recognition of these simple organic cations by MDR+ cells. Thus, MDR+ cells are not cross-resistant to the nonaromatic guanidiniums but do show cross-resistance to those aromatic pyridiniums with chain lengthsfour. Resistance ratios, as determined by comparison of 50% inhibitory doses in MDR- versus MDR+ cells, increase as a function of increasing chain lengths of these latter simple aromatic compounds. Resistance to pyridinium analogues in MDR+ cells is reversible by co-treatment with nontoxic doses of verapamil. Preliminary uptake data with radioactive analogues further implicate the MDR mechanism of lowered drug accumulation in accounting for resistance to the pyridinium homologues. Utilization of these simple organic cations provides a rational basis for better defining the physical chemical properties of more complex compounds processed by the MDR mechanism and suggests a strategy for designing chemotherapeutic agents with reduced susceptibility to MDR.

Tumor cell resistance to anthracyclines has been associated with increased activity against free radicals. Here, we have investigated the direct effect of doxorubicin (DOX) in the modulation of glutathione level and antioxidant activities in DOX-sensitive and-resistant cells (288 fold). The glutathione level in untreated cells was 88% greater in resistant than in sensitive cells. The activities of the superoxide dismutase, glutathione -S-transferase and glutathione reductase were respectively 24, 15 and 38% higher in resistant cells than in their sensitive counterparts. In contrast, catalase and total glutathione peroxidase were reduced in resistant cells by 18 and 21% respectively. Moreover, the activity of selenium-dependent glutathione peroxidase was lowered by 47% in the resistant as compared to the sensitive cells. Exposure of sensitive or resistant cells to low doses of DOX did not affect these levels in either cell variant. It is concluded therefore that resistance to anthracyclines may not always be associated with an elevated level of intracellular antioxidant activity enzymes.

The mechanism of action of 3'-deamino-3'-morpholino-13-deoxo-10-hydroxycarminomycin (MX2) was examined in a human leukemia cell line (K562) and its Adriamycin (ADM)-resistant subline (K562/ADM). ADM and MX2 showed an equivalent antitumor effect against K562. K562/ADM was highly resistant to ADM. In cellular pharmacokinetic studies, MX2 showed faster and greater influx than did ADM in both K562 and K562/ADM. The efflux of ADM was rapid in K562/ADM but not in K562. On the other hand, the efflux of MX2 was rapid in both cell lines. The formation of DNA single-strand breaks and double-strand breaks by ADM was significantly lower in K562/ADM than K562. On the other hand, formation of those breaks by MX2 was not decreased. Although some of the DNA breaks induced by MX2 were resealed, there was no difference in the degree of resealing in K562 and K562/ADM cells. On the other hand, most of the small number of DNA breaks in K562/ADM induced by ADM were resealed. The topoisomerase II activity in K562 and K562/ADM was not significantly different. It is concluded that MX2 conquers multidrug resistance by rapid influx following a higher frequency of formation of DNA single- and double-strand breaks in K562/ADM cells.

Accumulation, cytotoxicity, and DNA damages produced by doxorubicin (DOX), pirarubicin (THP-DOX), fluoro-doxorubicin (ME2303) or its isolated metabolite M1 have been investigated in human myelogenous leukemia cells, sensitive (K562) and resistant to DOX (K562/DOX). These compounds differed by lipophilicity and/or sugar moiety either with (DOX, THP-DOX) or without (ME2303, M1) amino group. In K562 cells, the cytotoxicity was correlated to DNA single-stranded breaks and the intracellular drug amount of DOX or M1. This was not true when the cells were treated with THP-DOX or ME2303. In addition, THP-DOX produced total DNA protein cross-linking. In K562 cells DNA damage was not repaired, while in K562/DOX repair of DNA damage produced by all drugs could be observed. Although in K562/DOX cells drug accumulation was much reduced, higher intracellular drug concentration was required to induce similar level of cytotoxicity and DNA damage. Thus, cytotoxicity produced by anthracycline is not always associated with DNA damage. Different level of resistance to DOX, THP-DOX, ME2303 or M1 is associated with reduced drug accumulation which varies with the structure.

Chick embryo fibroblasts in monolayer culture were synchronized by contact inhibition and serum starvation. Nuclear DNA isolated from the [ 3 H] thymidine pulse-labelled cells throughout the period of DNA synthesis (S phase) was analysed by hydroxylapatite chromatography after renaturation at different C 0 t values. It is shown that repeated sequences having different frequencies of reassociation, replicate differently throughout the S period. In order to study the distribution of the repeated sequences, DNA isolated during the S period was fractionated according to its buoyant density. It is shown that only some of the highly reiterated sequences which are included in the high buoyant density DNA fractions, replicate equally well during the early and the late S periods. By contrast, reiterated sequences of the low buoyant density DNA fractions replicate mainly during the late S period.

Diploid Chinese hamster cells were labelled with 32 P for 2 days or synchronized and pulse-labelled with 3 H-thymidine throughout the DNA synthetic (S) period. Total nuclear DNA and DNA fractions having different G + C contents were analysed, using an improved method of hydroxylapatite chromatography. Both uniformly labelled DNA (random DNA) and DNA pulse-labelled throughout the S period, contained a similar proportion (3–4%) of single-stranded sequences (SS-DNA). The amount and G + C content of SS-DNA were very much increased in the highest buoyant density DNA fraction analysed. After denaturing and renaturing at several C 0 t values, the amounts of reassociated DNA were determined. Using random DNA, the G + C content of the reassociated sequences did not differ from that of total DNA regardless of the C 0 t value. Using pulse-labelled DNA, the proportion of highly repetitious sequences (renatured at C 0 t 2) isolated from total DNA as well as from both high and low buoyant density fractions, did not vary throughout the S period, as compared with random DNA. However, both the heavy and light fractions of late S DNA contained a greater proportion of moderately repetitious sequences (renatured at C 0 t 10 and 50) than early S or random DNA. These results are compared with those in other animal species.

Friend leukemia cells were induced to differentiate by hexamethylene-bis-acetamide (HMBA). The relationship between cell growth cell volume distribution, fluorescence intensity and fluorescence anisotropy of the membrane probe 1,6 diphenyl 1,3,5 hexatriene (DPH) was analyzed in differentiated and undifferentiated cells with the aid of the fluorescence activated cell sorter (FACS II). The induction of a differentiated state was associated with a decrease in cell volume and an increase of fluorescence anisotrophy. The inhibition of this induction by dexamethasone prevented the decrease of cell volume without affecting the increase of fluorescence anisotropy. We conclude therefore that the high degree of anisotropy is not correlated to the differentiated state nor with the volume of the cells.

Changes in phospholipids and in fatty acid composition were observed when Friend leukemia cells (FLC), but not the resistant one (RFLC) were exposed to DMSO. As a result of these changes, the molar ratio of phosphatidyl choline (PC) to phosphatidyl ethanolamine (PE) and the relative amount of arachidonic acid (20:4) isolated from total lipids, PC and PE increased in differentiated cells. We therefore conclude that erythroid differentiation induced by DMSO is related to changes in the lipid components of FLC membranes.

In a previous study, using fluorescence polarization (P) with diphenyl hexatriene (DPH), it was shown that growing and resting cells have different P-values. This property has now been used to investigate the membrane action of dimethylsulfoxide (DMSO) and hexamethylene bisacetamide (HMBA), two inducers of Friend leukaemic cell (FLC) differentiation. Both an inducible cell line (FLC) and a resistant variant cell line (RFLC), had the same characteristics regarding P-values. During the differentiation process in the FLC cell line, further changes in P-values were observed. In the resistant cell line, these changes in P-value were not seen. This suggests that these inducers of differentiation are acting at the cell membrane.

Friend leukaemic cells (FLC) were induced to differentiate with dimethyl sulfoxyde (DMSO), hexamethylenbis-acetamide (HMBA) and sodium butyrate (SB) and the phospholipid composition was analyzed. The phospholipid composition of differentiated cells differed from that of non differentiated cells and also varied according to inducer. The ratios of the percentage of phosphatidyl choline (PC) to that of phosphatidyl ethanolamine (PE) or sphingomyelin (SPH) increased by about 2-fold in DMSO or SB induced FLC. These ratios did not vary in HMBa induced FLC. Furthermore the fatty acid composition of PC and PE obtained from differentiated cells varied according to the inducer. Although these changes appeared to be related to the inducers, it can not be excluded that the differentiated state also contributes to these changes.

The relationship between membrane dynamics and cellular growth of Friend leukaemic cells (FLC) was studied by fluorescence polarization (P) using diphenyl hexatriene (DPH). The P-value changes as a function of the state of the cells being maximum in the growing state. The P-value is also influenced by serum concentration and this effect appears to be a passive process.

Based on the fluorescence properties of adriamycin (ADM) and daunorubicin (DNR), uptake in sensitive and resistant Friend leukemia cells (FLC) was studied with the aid of the fluorescence activated cell sorter (FACS II). A quantitative cell by cell fluorescence intensity analysis showed a differential affinity of FLC to ADM and DNR. The cellular uptake of these two drugs was temperature dependent and was not hindered by sodium azide treatment; incorporation into isolated nuclei was not temperature dependent, nor hindered by sodium azide. Friend leukemia cell variants resistant to adriamycin (ADM-RFLC) and to daunorubicin (DNR-RFLC) were developed. The rate of uptake of ADM and DNR across the plasma membrane of these two cell variants was lower than in sensitive cells. Although these cells were crossresistant to both ADM and DNR, the drug-induced fluorescence intensity was distributed differently in the corresponding resistant cell variants. We suggest therefore that resistant is the consequence of changes induced in the plasma membrane components. These changes may differ according to which drug is used.