Quantitative study of natural antioxidant systems for cellular nitrofurantoin toxicity

The toxicity of nitrofurantoin was studied on human WI-38 fibroblasts: this chemical was lethal when added at concentrations higher than 5·10⁻⁵ M in the culture medium. The protection afforded by anitoxidants was then tested: α-tocopherol gave at 10⁻⁴ M a light protection in contrast to ascorbic acid which even became toxic at high concentrations. We also tested catalase, superoxide dismutase and glutathione peroxidase introduced intracellularly by the microinjection technique. On a molecular basis, glutathione peroxidase was 23-times more efficient than catalase and 3000-times more than superoxide dismutase. The results also showed that a similar range of enzyme concentrations was found for the protection against high oxygen pressure. This suggests that, in the case of both oxygen and nitrofurantoin toxicity, the peroxide derivatives are the most toxic intermediates of the free radical attacks.     

Mechanism of nitrofurantoin toxicity and oxidative stress in mitochondria

5-Nitrofuran derivatives change the inner mitochondrial membrane permeability as indicated by the transmembrane potential, the rate of spontaneous K+ efflux and the basal respiratory rate: (a) at low concentrations nitrofurantoin prevents the increase of inner membrane permeability due to hydroperoxides or to diamide; (b) at higher concentrations or after longer times of incubation, nitrofurantoin enhances the membrane damage due to hydroperoxides or to diamide; the damage due Ca2+ plus Pi is enhanced by nitrofurantoin at all concentrations; (c) higher nitrofurantoin concentrations cause membrane damage independently of the presence of hydroperoxides or of diamide. The effect of nitrofurantoin is cancelled by the addition of free-radical scavengers. The above effects of nitrofurantoin are compatible with the observations of Mason and colleagues that nitrofurantoin is reduced by a NADPH nitroreductase to a nitro anion radical which can then undergo subsequent reactions, among which are (a) initiation of a free-radical reaction chain and (b) reduction of hydroperoxides and diamide.     

Quantitative inference of cellular parameters from microfluidic cell culture systems

Microfluidic cell culture systems offer a convenient way to measure cell biophysical parameters in conditions close to the physiological environment. We demonstrate the application of a mathematical model describing the spatial distribution of nutrient and growth factor concentrations in inferring cellular oxygen uptake rates from experimental measurements. We use experimental measurements of oxygen concentrations in a poly(dimethylsiloxane) (PDMS) microreactor culturing human hepatocellular liver carcinoma cells (HepG2) to infer quantitative information on cellular oxygen uptake rates. We use a novel microchannel design to avoid the parameter correlation problem associated with simultaneous cellular uptake and diffusion of oxygen through the PDMS surface. We find that the cellular uptake of oxygen is dependent on the cell density and can be modeled using a logistic term in the Michaelis–Menten equation. Our results are significant not only for the development of novel assays to quantitatively infer cell response to stimuli, but also for the development, design, and optimization of novel in vitro systems for drug discovery and tissue engineering. Biotechnol. Bioeng. 2009;103: 966–974. © 2009 Wiley Periodicals, Inc.     

In vitro microscale systems for systematic drug toxicity study

After administration, drugs go through a complex, dynamic process of absorption, distribution, metabolism and excretion. The resulting time-dependent concentration, termed pharmacokinetics (PK), is critical to the pharmacological response from patients. An in vitro system that can test the dynamics of drug effects in a more systematic way would save time and costs in drug development. Integration of microfabrication and cell culture techniques has resulted in ‘cells-on-a-chip’ technology, which is showing promise for high-throughput drug screening in physiologically relevant manner. In this review, we summarize current research efforts which ultimately lead to in vitro systems for testing drug’s effect in PK-based manner. In particular, we highlight the contribution of microscale systems towards this goal. We envision that the ‘cells-on-a-chip’ technology will serve as a valuable link between in vitro and in vivo studies, reducing the demand for animal studies, and making clinical trials more effective.     

Interrelations of natural cellular cytotoxicity and interferon systems in immobilization stress

The suppressed cytotoxic natural killer activity was observed in the mouse spleen during 2 days after 6 h of immobilization stress. Both serum interferon level and interferon production by spleen cells under PHA, ConA and enterotoxin stimulation were also significantly decreased. The period of suppression was followed by the recovery of the activity of the two systems by day 7-9. The experimental data form the basis for the suggestion that endogenous interferon has major significance for the mechanism of disturbed natural killer activity after stress influences.     

Oxalic acid is available as a natural antioxidant in some systems

Oxalic acid is found in a wide variety of plants. This study showed that oxalic acid suppressed in vitro lipid peroxidation in a concentration-dependent manner. Furthermore, oxalic acid reduced the rate of ascorbic acid oxidation in the presence of hydrogen peroxide and Cu(2+). These results suggest that oxalic acid is available as a natural antioxidant.     

Biomimetic membrane systems to study cellular organization

During many cellular processes such as cell division, polarization and motility, the plasma membrane does not only represent a passive physical barrier, but also provides a highly dynamic platform for the interplay between lipids, membrane binding proteins and cytoskeletal elements. Even though many regulators of these interactions are known, their mutual interdependence appears to be highly complex and difficult to study in a living cell. Over the past few years, in vitro studies on membrane–cytoskeleton interactions using biomimetic membranes turned out to be extremely helpful to get better mechanistic insight into the dynamics of these processes. In this review, we discuss some of the recent developments using in vitro assays to dissect the role of the players involved: lipids in the membrane, proteins binding to membranes and proteins binding to membrane proteins. We also summarize advantages and disadvantages of supported lipid bilayers as model membrane.     

Quercetin handles cellular oxidant/antioxidant systems and mitigates immunosenescence hallmarks in human PBMCs: An in vitro study

Immunosenescence, oxidative stress, and low vaccine efficacy are important symptoms of aging. The goal of our study was to test if quercetin had antiaging and stimulating effects on peripheral blood mononuclear cell (PBMC) immune cells in vitro in the presence of concanavalin a, PBMCs were isolated from healthy elderly and young people and cultured in a complete Roswell Park Memorial Institute 1640 medium supplemented with quercetin. Cell proliferation was assessed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide colorimetric assay after a 48-h incubation period. Spectrophotometric assays were used to assess oxidative biomarkers (proteins carbonyl [PC], malondialdehydes [MDA], and reduced glutathione [GSH]). The enzyme-linked immunosorbent assay method was used to determine the amount of interleukin (IL)-2 released. A Griess reagent was used to investigate inducible nitrite oxide synthase (iNOS) activity. When compared to young control cells, aged PBMCs had lower proliferation potency, lower IL-2 and NO release, and higher MDA and PC levels. Importantly, quercetin-treated aged PBMCs have a high proliferative response comparable to young cells, restored iNOS activity, and increased levels of GSH antioxidant defences. In comparison to untreated aged PBMCs, treated PBMCs have lower lipo-oxidative damage but higher PC levels. Quercetin may be used as a promising dietary vaccinal adjuvant in the elderly, it has significant effects in reducing immunosenescence hallmarks, as well as mitigating the lipo-oxidative stress in PBMCs cells.     

Quantitative risk assessment for developmental toxicity.

Pharmaceutical companies and governmental regulatory agencies are becoming increasingly aware of the need for improved statistical methods for developmental toxicity experiments. Although a number of statisticians have become interested in this area, activity has centered mostly on the development of methods to analyze binary outcomes, such as malformations among live pups, while accounting appropriately for the correlation induced by the litter effect. In contrast, the topic of quantitative risk assessment has received relatively little attention. This paper addresses the specific question of how to assess risk appropriately when exposure causes a variety of adverse effects, including resorption and fetal death, in addition to malformations. It will be seen that risk assessments based on a single developmental outcome, such as malformation, may be conservative. A method is proposed for estimating an exposure level at which the overall risk of any adverse effect is acceptably low. The method is based on a continuation ratio formulation of a multinomial distribution, with an additional scale parameter to account for overdispersion. Comparisons are made with binary models on prenatal death and malformation, as well as a binary model that makes no distinction between death and malformation, but simply classifies each fetus as normal or abnormal. Data from several developmental toxicity studies illustrate the results and findings.     

Expression of genes that encode cellular oxidant/antioxidant systems are affected by heat stress

Molecular Biology Reports - Heat stress causes critical molecular dysfunction that affects productivity in chickens. Thus, the purpose of this study was to evaluate the effect of heat stress (HS)...     

Quantitative aspects of cellular turnover

Living organisms do not just grow by synthesizing cellular components. As part of the necessary steps for existence, some components are degraded after synthesis. Even for bacteria in balanced, exponential growth some substances, under some conditions, are turned over. In other phases of growth turnover can be much more extensive, but it is still selective. This review covers studies with animals as a way to put the studies on microorganisms in perspective. The history, the mathematics, and experimental design of turnover experiments are reviewed. The important conclusion is that most of the proteins during balanced growth are very stable in bacteria, although ribosomal proteins are degraded under starvation conditions. Another generalization is that the process of wall enlargement in general is associated with obligatory turnover of the peptidoglycan.     

The cellular toxicity of aluminium.

Aluminium is a serious environmental toxicant and is inimical to biota. Omnipresent, it is linked with a number of disorders in man including Alzheimer's disease, Parkinson's dementia and osteomalacia. Evidence supporting aluminium as an aetiological agent in such disorders is not conclusive and suffers principally from a lack of consensus with respect to aluminium's toxic mode of action. Obligatory to the elucidation of toxic mechanisms is an understanding of the biological availability of aluminium. This describes the fate of and response to aluminium in any biological system and is thus an important influence of the toxicity of aluminium. A general theme in much aluminium toxicity is an accelerated cell death. Herein mechanisms are described to account for cell death from both acute and chronic aluminium challenges. Aluminium associations with both extracellular surfaces and intracellular ligands are implicated. The cellular response to aluminium is found to be biphasic having both stimulatory and inhibitory components. In either case the disruption of second messenger systems is observed and GTPase cycles are potential target sites. Specific ligands for aluminium at these sites are unknown though are likely to be proteins upon which oxygen-based functional groups are orientated to give exceptionally strong binding with the free aluminium ion.     

Molecular bases of cellular iron toxicity

Patients with hereditary or secondary hemochromatosis are liable to cardiac and hepatic failure, and type II diabetes. Despite the highly likely conjecture that iron-mediated tissue damage involves the conspiracy of cellular oxidizing and reducing equivalents, the pathophysiologic events have not been fully elucidated. These latter likely involve toxic effects of iron on intracellular organelles, in particular, mitochondria and lysosomes. The tissues at risk-heart, liver, and pancreatic beta cells-all have highly active mitochondria, which incidentally generate activated oxygen species capable of causing synergistic toxicity with intracellular iron. This suggests the general concept that iron may be preferentially toxic to cells with high mitochondrial activity. At least part of the long-term toxicity may involve iron-mediated oxidative damage to the mitochondrial genome with an accumulation of mutational events leading to progressive mitochondrial dysfunction. An alternative-and not mutually exclusive-mechanism for cellular iron toxicity involves iron-catalyzed oxidative destabilization of lysosomes, leading to leak of digestive enzymes into the cell cytoplasm and eventuating in apoptotic or necrotic cell death.     

Induction of 6-thioguanine-resistant mutants by hyperoxia and gamma-irradiation: effect of compromising cellular antioxidant systems

Hyperoxia and gamma-irradiation were found to be mutagenic in a transformed Syrian hamster cell line in a dose-dependent manner. The frequency of resistance to 6-thioguanine increased from 10 per 10(6) survivors after 48 h of growth in 70% O2 to 32.6 (highly significant) after 75 h. Increasing the oxygen tension to 95% resulted in a significant mutagenic response in only 44 h. At equitoxic doses, gamma-irradiation was 4 times more mutagenic than 70% O2. After growth in hyperoxia, the cells showed an enhancement of catalase activity, glutathione peroxidase activity and glutathione levels but there was little effect on superoxide dismutase activity. Diethyldithiocarbamate (3 mM, 1.5 h) was mutagenic in normoxia and potentiated the mutagenic activity of both gamma-irradiation and hyperoxia. Cells thus treated showed an 855 reduction in superoxide dismutase activity. When diethyldithiocarbamate was used in conjunction with a direct-acting alkylating agent, the mutagenic response was only additive. Depletion of cellular glutathione with buthionine sulfoximine (0.2 mM) or inhibition of catalase activity with aminotriazole (100 mM) was also effective in potentiating the mutagenic response of gamma-irradiation and hyperoxia. The data demonstrates that endogenously produced activated oxygen species are mutagenic to hamster cells in culture and suggest that aerobic organisms are subject to an unavoidable background risk due to living in an oxygen atmosphere.     

Lipid peroxidation and antioxidant systems in the blood of young rats subjected to chronic fluoride toxicity

Wistar albino rats were exposed to 30 or 100 ppm fluoride in drinking water during their fetal, weanling and post-weaning stages of life up to puberty. Extent of lipid peroxidation and response of the antioxidant systems in red blood cells and plasma to prolonged fluoride exposure were assessed in these rats in comparison to the control rats fed with permissible level (0.5 ppm) of fluoride. Rats treated with 100 ppm fluoride showed enhanced lipid peroxidation as evidenced by elevated malondialdehyde (MDA) levels in red blood cells but, 30 ppm fluoride did not cause any appreciable change in RBC MDA level. 30 ppm fluoride-intake resulted in increased levels of total and reduced glutathione in red blood cells and ascorbic acid in plasma while 100 ppm fluoride resulted in decreases in these levels. The activity of RBC glutathione peroxidase was elevated in both the fluoride-treated groups, more pronounced increase was seen with 100 ppm. Reduced to total glutathione ratio in RBC and uric acid levels in plasma decreased in both the groups. RBC superoxide dismutase activity decreased significantly on high-fluoride treatment. These results suggest that long-term high-fluoride intake at the early developing stages of life enhances oxidative stress in the blood, thereby disturbing the antioxidant defense of rats. Increased oxidative stress could be one of the mediating factors in the pathogenesis of toxic manifestations of fluoride.     

Some natural viability systems for a multiallelic locus: a theoretical study

The maintenance of genetic polymorphism under various natural structured viability regimes vs. general unrestricted fitness assignments are compared. The selection models considered include a generalized dominance fitness system, a generalized viability model based on allelic activity values, viability matrices based on multilocus activity levels, viability matrices defined by partitioned "resource" or "substrate" variables, and circulant-type viability matrices. A number of examples that support these formulations are discussed. Detailed results on the nature of the genotype frequency equilibrium configurations for the specified viability models are presented. An increased likelihood for a globally stable equilibrium is predicted for the more structured viability models.