After 24, 48, and 72 h of incubation, an aliquot of the cells was stained with propidium iodide and subjected to flow cytometric analysis to assess DNA configuration. Phytohaemagglutinin stimulation led to a significant increase in the S phase of the cell cycle after 48 and 72 hours of incubation.
1. Introduction
Literature review .1 Mycotoxins
- White blood cells
- Lymphocyte function and ultrastructural features
- Other white cells .1 The monocytes
- Neutrophils
- Eosinophils
- Basophils
- Proteins as molecular receptors
- Effects of mycotoxins on cellular respiration
Basophils (Fig. 5) are the least abundant leukocytes, constituting approximately 1% of the leukocytes in the circulating blood. ATP depletion can then cause mitochondrial swelling due to the cell's inability to maintain sodium and potassium gradients.
PICOLINIC ACID
- Cytotoxic effect of picolinic acid
- Antitumour Activity
It is also known to act synergistically with IFNγ in activating macrophages, leading to the expression of tumoricidal activity by macrophages (Varesio et al., 1990). The results suggest that the process of activation of tumoricidal macrophages also initiates a mechanism of resistance to viral mRNA expression (Varesio et al., 1990).
Fusaric acid (5-hutylpicolinic acid)
- The effect of fusaric acid on animals
- The relation of fusaric acid to niacin
- Apoptosis and cytotoxins
- BcI-2
There are essentially two phases to the response. i) the first involves events at the receptor site, which may include the degree of inhibition of a specific enzyme or the degree of DNA damage. ii) the second involves the homeostatic adaptation of cells to the resulting stimulus. Caspase cleavage of nuclear lamins results in nuclear shrinkage and budding (Buendia et al., 1999).
Apoptosis and the mitochondria
- The formation of channels by the BcI-2 family to facilitate protein transport It has been suggested that the bcl-2 proteins might act by inserting, following
- The interaction of BcI-2 with other proteins to form channels
- BcI-2 family members may cause rupture ofthe mitochondrial membrane Mitochondrial homeostasis may be under the control of bcl-2 family members
The release of pro-apoptotic factors from mitochondria does not necessarily result in apoptotic cell death. These anti-apoptotic bcl-2 family members can be viewed as buffers that minimize the accidental release of mitochondrial contents (Hengartner, 2000).
CD95 and apoptosis
One of the F ADD domains is the death effector domain (DED) and through a homologous interaction process, it recruits the DED-containing procaspase-8 (also known as FLICE) to the DISC (Krammer, 2000). One of the main targets of active caspase-8 is procaspase 3, which initiates the activation and execution of the complete cell death program (Krammer, 2000).
Cytotoxicity testing: Methyl thiazole tetrazolium assay
- Materials and method .1 Materials
- Cell culture media
- Methods
- Results and Discussion
- Determination of Genotoxicity
- Statistical analysis
- Results and Discussion
The sensitivity of the comet assay in assessing DNA damage depends on the accurate and reproducible measurement of DNA in the head and tail regions of the comet. The sensitivity of the SCGE assay extends to the detection of less than 0.1 breaks per 109 daltons of DNA (Gedik et al., 1992). The most widely used version of the SCGE assay is that of Singh et al., (1988) in which electrophoresis is performed at high pH.
The properties of the SCGE assay highlight it as an assay of DNA breaks with widespread applications in DNA damage and repair studies, biomonitoring, genetic toxicology, and analysis of irradiated foods. Thus, the results of DNA damage induced by FAin human lymphocytes are significantly different from those of controls. Lymphocytes showed significant increase in DNA damage at all concentrations of FA after 1 h of incubation.
The DNA damage induced by FA, whether endogenous or exogenous, can be easily detected by the SCGE assay.
DNA Fragmentation analysis
- The role of endonuclease in DNA fragmentation
- Electrophoresis
- Results and Discussion
- Materials and Method .1 Materials
- Methods
- Results and Discussion
- Nuclear envelope and nuclear pores
The analysis of transmission electron microscopy is considered a historical moment of research in the field of apoptosis. Chromatin associated with the nucleus has focal aggregates of chromatin granules along the periphery of the nucleus. This is the most characteristic form of swelling, ie swelling of the inner chamber containing the mitochondrial matrix (Fig 29) (Ghadially, 1982).
The outer membrane of the nuclear envelope is often studded with few or many ribosomes. A common occurrence is the fusion of two membranes in the nuclear envelope, producing 'fenestrations' called the nuclear pores (Fig. 34). Cristae (black arrows) are displaced and almost exclusively located in the periphery of the mitochondria.
The most common form of swelling is that caused by the involvement of the matrix or inner chamber.
Detection of apoptotic cells by light scatter analysis
- Apoptotic assessment/quantification by detection of phosphatidylserine with Annexin V-FITC conjugate
The timely generation of recognition signals on the surface of apoptotic cells is a key event in the apoptotic program (Verhoeven et al., 1995). Phospholipids found within the plasma membrane are asymmetrically distributed between the inner and outer leaflets of the plasma membrane. In living cells, phosphatidylserine (PS) is observed almost exclusively on the inner surface of the membrane.
Recently, loss of phosphatidyl asymmetry, leading to exposure of PS on the outside of the plasma membrane, has been shown to be an early event in apoptosis. Annexin, when conjugated to a fluorochrome, can be used as a sensitive probe for the presence of PS on the outside of the plasma membrane (Fig. 38). The translocation of PS to the outer membrane is not exclusive to apoptosis but also occurs during necrosis.
During apoptosis, cells take up Annexin V after the onset of chromatin condensation, but before the loss of the ability of the plasma membrane to exclude cationic dyes such as PI (Vermes et al., 1995; Koopman et al., 1994) .
Annaxln V-FI"
The role of phosphatidylserine in apoptosis
- Aminophospholipid translocase
- ATP-dependent floppase
- Lipid Scramblase
- Implications of the annexin structure
The outer membrane of the eukaryotic cell contains mainly cholinephospholipids (sphingomyelin and phosphatidylcholine [PC]), while most aminophospholipids (phosphatidylserine [PS] phosphatidylethanolamine [PE]) are confined to the membrane. These vesicles were found to be capable of translocating a spin-labeled PS analog from the inner to the outer leaflet of the membrane upon addition of Mg2+-ATP. This less specific ATP-requiring flopase transports aminophospholipids and cholinephospholipids from the inner to the outer compartment with half-times about 10 times longer than those of the translocase-mediated inward movement of PS and PE.
The basic structure of the annexins consists of four or eight repeats of 70 amino acids in length. Each of the 70 amino acid domains in the annexin protein forms a bundle of five, nearly parallel (or antiparallel), alpha helices wound into a right-handed superhelix. The calcium binding sites are all found on one side of the planar annexin molecule.
This forms a clip that attaches the first domain to the fourth domain and thus maintains the circular arrangement of the 70 amino acid domains (Verheij et al., 1980).
Materials and Method .1 Materials
- Methods
- Preparation of lymphocyte samples for flow cytometry
- Flow cytometric analysis
- Data analysis
With regard to annexin ii, where the p 1 0 binding site near the amino terminus of the heavy chain presumably faces the cytoplasm, like the amino terminus of annexin V, it is possible that the two heavy chains have opposite orientations and therefore can bind two approaching membranes simultaneously. A high degree of ion selectivity is provided by an ion permeation route through the hydrophilic central channel of the protein (Pollard and Rojas, 1988). Lymphocyte suspensions were treated with a series of serial dilutions and supplemented to final concentrations of 3 µM, 6 µM, 25 µM, 50 µM, 200 µM and 400 µM FA.
At each respective time interval, lymphocytes (106 cells/ml) were collected by centrifugation at 400xg for 5 minutes. Lymphocytes were then treated with 100 µl of staining solution and incubated for 15 minutes at 22°C to allow the cells to acquire the appropriate stains. Lymphocytes were then analyzed on a Becton Dickenson Facs Calibur flow cytometer using a 488 nm excitation and 515 nm bandpass filter for fluorescein detection and a greater than 560 nm filter for PI detection.
Electronic compensation of the instrument was performed to exclude overlap of the two emission spectra (appendix 6).
Results and Discussion
Values are given as the mean percentages of three experiments ± the standard error of the mean. Quantification of apoptosis by microscopy is cumbersome, while most fluorescent dyes used in flow cytometry are nonvital dyes, that is, they only stain apoptotic cells after membrane damage. This population of double-stained lymphocytes was also significantly higher compared to those from the 1 and 4 hour incubations.
The versatility of flow cytometry (FC) in a number of methodologies applicable to individual cells or cell organelles has more recently led to its widespread use in studies of cell proliferation (cell cycle). The use of FC in proliferation assays serves two main purposes: 1) revealing distribution of cells in particular phases of the cycle or determining the kinetics of progression through these phases; 2) the elucidation of molecular and functional mechanisms associated with the cell cycle. If asynchronously growing cells are treated in vitro or in vivo with an agent that interferes with progression through the cell cycle, cells tend to accumulate in the phase where their progression is maximally affected, shown as an increase in relative proportion of cells in that phase of the cycle on the DNA content frequency histograms (Darzynkiewicz et al., 2001). A disadvantage with frequency histograms is that they do not provide information about the rate of cell cycle progression or kinetics.
For these purposes, the duration of the cell cycle, or the doubling time of cells in culture, is required (Darzynkiewicz et al., 2001).
DNA Content
Results and Discussion
Significant changes in the different phases due to co-incubation with FA occurred predominantly after 48 and 72 h. Frequency histograms (Figs. 48 a-c, 49a-c and 50 a-c) illustrate changes in the different phases at different time intervals and concentrations. Aflatoxin exposure and DNA damage in the comet assay in individuals from The Gambia, West Mrica, Teratogenesis, Carcinogenesis and Mutagenesis.
Peripheral and short-term effects of fusaric acid, a DBH inhibitor, on tryptophan and serotonin metabolism in the rat, Journal of Neural Transmission. Asymmetric distribution of spin-labeled phospholipids in the erythrocyte membrane: Relation to shape changes, Proceedings of the National Academy of Sciences USA. Requirements for phosphatidylinositol14,5-bisphosphate in the Ca2+-induced phospholipid redistribution in the human erythrocyte membrane, Journal of Biological Chemistry.
Picolinic acid, a catabolite of tryptophan, as the second signal in the activation of IFN-γ-primed macrophages, The Journal of Immunology.
Lysis buffer
Lysing solution
While viewing an FSC versus SSC dot plot, run the control tube with unstained cells and set the acquisition gate to the population of interest. Adjust the emission PMTs so that >98% of the total events are in the lower left quadrant and within the first log decade on both the X and Y axes of the FL1 versus FL2 (or FL3) dot plot. While viewing the FL1 versus FL2 (or FL3) dot plot of the gated cells, run tube #2 (Appendix V-FITC only) to ensure that no events are recorded in the upper left and upper right quadrants of the screen.
While viewing the FL1 vs FL2 (or FL3) dot plot of the gated cells, run tube #3 (PI only) to ensure that no events are detected in the upper and lower right quadrants of the screen. If PI-stained cells are visible in other than the upper left quadrant, reduce the FL1 - %FL2 (or FL3) compensation (this can vary from 0-1%). If the flow cytometer has been properly compensated, single-stained cells should be centrally located in the upper left (FL2 or FL3IPI) or lower right (FLlIannexin V-FITC) quadrants.
To properly set the quadrant markers for determining the frequency of cells undergoing apoptosis, FL1 vs FL2 (or FL3).
