Cell Membrane Lysis Research Articles

부평초약침이 지방세포 대사에 미치는 영향

Objectives : The purpose of this study is to investigate the effects of Spirodelae Herba pharmacopuncture(SHP) on the adipogenesis in 3T3 cells, lipolysis in rat epididymal adipocytes and histological changes in porcine adipose tissue. Methods : Inhibition of preadipocyte differentiation and/or stimulation of lipolysis play important roles in reducing obesity. 3T3 preadipocytes were differentiated with adipogenic reagents by incubating for 3 days in the absence or presence of SHP ranging from 0.01 to . The effect of SHP on adipogenesis was examined by measuring glycerol-3-phosphate dehydrogenase(GPDH) activity and by Oil Red O staining. Mature adipocytes from rat epididymal fat pad was incubated with SHP ranging from 0.01 to for 3 days. The effect of SHP on lipolysis was examined by measuring free glycerol released. Fat tissue from porcine skin was injected with SHP ranging from 0.1 to to examine the effect of SHP onhistological changes under light microscopy. Results : Following results were obtained from the preadipocyte proliferation and lipolysis adipocyte and histologic investigation of fat tissue 1. SHP showed the effect of decreased preadipocyte proliferation on the high dosage(). 2. SHP showed the effect of decreased the activity of glycerol-3-phosphate dehydrogenase (GPDH) on the high dosage(). 3. Investigated the changes in lipolysis of differentiated adipocyte after treated SHP, we knew that these pharmacopuncture showed increasing the effect of lipolysis in all concentration significantly. 4. Investigated the histological changes in porcine fat tissue after treated SHP, we knew that these pharmacopuncture showed significant activity to the lysis of extensive cell membranes on high dosage(). Conclusions : These results suggest that SHP efficiently induces diminishing proliferation of preadipocyte and lipolysis in adipose tissue.

추출방식에 따른 방풍통성산(防風通聖散)이 지방세포 대사에 미치는 영향

Objective : The purpose of this study is to investigate the effects of Bangpungtongsung-san extracts on the preadipocytes proliferation, of 3T3-L1 cell line. lipolysis of adipocytes in rat`s epididymis and localized fat accumulation of porcine by extraction methods(alcohol and water). Methods : Diminish 3T3-L1 proliferation and lipogenesis do primary role to reduce obesity. So, 3T3-L1 preadipocyte and adipocytes were performed on cell cultures, and using Sprague-Dawley rats for the lipogenesis, and treated with 0.01- Bangpungtongsung-san Extracts depend on concentrations. Porcine skin including fat tissue after treated Bangpungtongsung-san Extracts by means of the dosage dependent variation are investigated the histologic changes after injection of these extracts. Results : Following results were obtained from the 3T3-L1 preadipocyte proliferation and lipolysis of adipocyte in rats and histologic investigation of fat tissue. 1. Bangpungtongsung-san extracts were showed the effect of decreased preadipocyte proliferation on the high dosage(). 2. Bangpungtongsung-san extracts were showed the effect of decreased the activity of glycerol-3-phosphate dehydrogenase(GPDH) on the high dosage() and Specially, alcohol extract of Bangpungtongsung-san was clear as time goes by high concentration. 3. Bangpungtongsung-san extracts were showed tries to compare the effect of lipolysis, alcohol extract of Bangpungtongsung-san on the high dosage() was observed the effect is higher than water extract. 4. Investigated the histological changes in porcine fat tissue after treated Bangpungtongsung-san extracts, we knew that water extract of Bangpungtongsung-san was showed the effect of lipolysis on the high dosage() and alcohol extract of Bangpungtongsung-san was showed significant activity to the lysis of cell membranes in all concentration. Conclusion : These results suggest that Bangpungtongsung-san efficiently induces diminish proliferation of preadipocyte and lipolysis in adipose tissue.

Tissue-Toxic Effects of Phosphatidylcholine/Deoxycholate after Subcutaneous Injection for Fat Dissolution in Rats and a Human Volunteer

The safety of the lipodissolution procedure for the cosmetic treatment of fat is unknown. The objective was to determine the subcutaneous tissue effects of phosphatidylcholine solubilized with deoxycholate (PC/DC) in rats and a human volunteer. Rats were treated subcutaneously three times with 50, 300, or 600 microL of PC/DC formula on the abdomen in a chronic study (30 days). A human volunteer undergoing elective liposuction was similarly treated. Cell membrane lysis, cell viability, and histologic status were determined on fresh biopsies of subcutaneous fat from the injection sites. PC/DC dose-dependently reduced membrane integrity and cell viability. Histologic alterations induced by PC/DC included fibroplasia, bandlike fibrosis in the region of the cutaneous muscle, and partial muscle loss. The highest dose caused widespread fat necrosis, fat cyst formation, and necrotic changes of the walls of small blood vessels. Histologic sections of subcutaneous tissue from the human volunteer showed dose-dependent panniculitis, fat cysts, and vessel necrosis. DC (2.5%), tested for comparison in the rat, exerted membrane and histologic effects similar to those of PC/DC. Solvent controls caused negligible alterations. Injection lipolysis with PC/DC causes tissue fibrosis and necrosis of adipose and vascular tissues in rat and man, making the long-term safety of PC/DC for nonsurgical treatment of subcutaneous fat deposits uncertain.

In Vitro Effects of Preservatives in Nasal Sprays on Human Nasal Epithelial Cells

The preservatives benzalkonium chloride and potassium sorbate are widely used in nasal drops and sprays. Recently, side effects resulting from mucosal damage caused by benzalkonium chloride and potassium sorbate were reported. We investigated the toxicity of benzalkonium chloride and potassium sorbate on human nasal epithelial cells in vitro. Using primary human nasal epithelial cells, different concentrations of benzalkonium chloride, potassium sorbate, or phosphate-buffered saline (PBS; control group) solutions were cocultured with nasal epithelial cells for 15 minutes. Then, the viability of the cells and the cell morphology were assessed. Nasal epithelial cells were more severely damaged with use of clinical preparations or higher concentrations of benzalkonium chloride than in the control group. In addition, nasal epithelial cell membrane lysis was seen on electronic microscopy in the benzalkonium chloride groups. In contrast, there was no significant cell damage seen in the potassium sorbate groups compared with the control group, even with higher concentrations than clinically used. Potassium sorbate appears to be a relatively safer preservative than benzalkonium chloride for use in nasal sprays and drops in vitro study.

Structure–activity relationships for the impact of selected isothiazol-3-one biocides on glutathione metabolism and glutathione reductase of the human liver cell line Hep G2

To investigate the toxic mode of action of isothiazol-3-one biocides the four compounds N-methylisothiazol-3-one (MIT), 5-chloro- N-methylisothiazol-3-one (CIT), N-octylisothiazol-3-one (OIT) and 4,5-dichloro- N-octylisothiazol-3-one (DCOIT) were purified and tested as single chemical entities for their effects on the human hepatoblastoma cell line Hep G2 and on isolated and cellular glutathione reductase GR). The two chlorinated substances CIT and DCOIT significantly decreased the amount of total cellular glutathione (GSx) in a dose and time dependent manner. Concomitantly, an increase in the level of oxidised glutathione (GSSG) was observed. The resulting shift in the GSH/GSSG ratio entailing the breakdown of the cellular thiol reduction potential was accompanied by necrotic morphological changes like swelling of the plasma membrane and subsequent lysis of the cells. Additionally, CIT and DCOIT were found to inhibit cellular GR in the cells in a concentration dependent manner. The T-SAR-based (thinking in terms of structure–activity relationships) comparison of the chlorine-substituted structures CIT and DCOIT with their non-chlorinated and less active analogues MIT and OIT identified the chlorine substituents and the resulting reaction mechanisms to be the key structural mediators of the observed toxic effects. Furthermore, differences in the activity of both chlorinated substances could be explained using the T-SAR approach to link the lipophilicity and the intrinsic glutathione-reactivity of the compounds to the expected target site concentrations inside the cells.

Bee Venom과 Sweet Bee Venom이 지방세포 대사에 미치는 영향

Objectives The purpose of this study was to investigate the effects of Bee Venom and Sweet Bee Venom to the primary cultured preadipocyte, adipocytes, and localized fat tissue. Methods Decreased preadipocyte proliferation and decreased lipogenesis are mechanisms to reduce obesity. So, preadipocytes and adipocytes were performed on cell cultures using Sprague-Dawley Rats and treated with 0.01-1mg/ml Bee Venom and Sweet Bee Venom. And porcine skin including fat tissue after treated Bee Venom and Sweet Bee Venom according to the dosage dependent variation are investigated the histologic changes after injection of these Pharmacopuncture. Result Following results were obtained from the preadipocyte proliferation and lipolysis of adipocyte and histologic investigation of fat tissue. 1. Bee Venom and Sweet Bee Venom showed the effect of decreased preadipocyte proliferation depend on concentration. 2. Bee Venom and Sweet Bee Venom showed the effect of decreased the activity of glycerol-3-phosphate dehydrogenase(GPDH) significantly. 3. Bee Venom was not showed the effect of lipolysis, but Sweet Bee Venom was increased in low dosage and decreased in high dosage. 4. Investigated the histologic changes in porcine fat tissue after treated Bee Venom and Sweet Bee Venom, we knew that these Pharmacopuncture was activated nonspecific lysis of cell membranes depend on concentration. Conclusion These results suggest that Bee Venom and Sweet Bee Venom efficiently induces decreased proliferation of preadipocyte and lipolysis in adipose tissue.

Exposure of Dunaliella tertiolecta to Lead and Aluminum: Toxicity and Effects on Ultrastructure

The growth response of the marine alga Dunaliella tertiolecta to different concentrations of lead and aluminum was investigated. Both metals had a stimulatory effect at low concentration and an inhibitory effect at high concentration (hormesis). The IC25 values of lead are 8.43, 7.29, and 6.74 mg L-1 for 24, 48, and 72 h, respectively. The corresponding values for aluminum are 30.54, 22.42, and 18.16 mg L-1. Although it seems that the two metals are not directly toxic to the alga at the concentrations found in the environment, as implied by the IC25 values and the environmental concentrations of the metals, low concentrations of both metals, alone and in combination, affected the ultrastructure. The growth of batch-grown cells exposed to 0.5 mg L-1 lead and aluminum, alone and combined, during the 24-h exponential phase was investigated. The same cells were also examined under an electron microscope to determine the biological effects of the two metals on the ultrastructure. The most obvious effects of lead were disrupted thylakoidal membranes, accumulated polyphosphate bodies and vacuoles, and lead precipitates on the cell surface. These ultrastructural alterations were partially present in aluminum-treated and lead-aluminum-treated cells. In joint exposure, the most important change was the lysis of the cell membrane. Aluminum and lead seem to act synergistically on the cell membrane leading to cell membrane lysis.

The confounding effects of light, sonication, and Mn(III)TBAP on quantitation of superoxide using hydroethidine

Previously, we showed that hydroethidine (HE) reacts with intracellular superoxide radical anion (O 2 −) to form a unique fluorescent marker product, 2-hydroxyethidium cation (2-OH-E +), that was not formed from HE reaction with other biologically relevant oxidants (H. Zhao et al. Proc. Natl. Acad. Sci. USA 102:5727–5732; 2005). Here we rigorously assessed the confounding effects of light, sonication, and Mn(III)TBAP on 2-OH-E +, the HE/O 2 − reaction product. Results indicate that continuous exposure to visible light induced photo-oxidation of HE to ethidium cation (E +) by a 2-OH-E +-dependent mechanism. Treatment of HE with ultrasound, a frequently used technique to lyse cell membranes, induced 2-OH-E + from in situ generation of O 2 −. Mn(III)TBAP, a cell-permeable metal–porphyrin complex used as a catalytic antioxidant, reacts with HE to form E +. This finding provides an alternative interpretation for Mn(III)TBAP effects during the HE/O 2 − reaction. In order to correctly interpret the HE reaction with O 2 − in cells, it is therefore imperative that HE and HE-derived products be measured by HPLC. A new and improved HPLC-electrochemical (HPLC-EC) detection has been developed for analysis of intracellular O 2 −. The HPLC-EC method is at least 10 times more sensitive than the HPLC-fluorescence technique for detecting O 2 − in cells.

Comparative study of red blood cell method in rat and calves blood as alternatives of Draize eye irritation test

Introduction Red blood cell assay (RBC) is used to estimate potential irritation of tensioactive agents and detergents. Cell membrane lysis and cell protein denaturation are measured photometrically. This study was aimed to determine if rat blood cells can be used to predict eye potential irritation in the same way of calves blood cells in RBC assay. Methods We evaluated 20 cosmetic formulations using rat and calves blood according to INVITOX protocol N° 37. Data of media hemolysis concentration, denaturation index and the ratio of both parameters were compared with in vivo data of eye irritancy. Results There was a significant difference ( p < 0.01) between H 50 value when evaluated the standard SDS with red blood cell method in rat and calves blood. According to the exact probability of Fisher taking as approach the acceptance or rejection of the substance there are no significant differences between in vitro assay with calves blood and in vivo results. Not happening the same way for the RBC assay with rat blood where significant differences were obtained ( p < 0.01) among the classification of in vitro and in vivo test. Discussion The RBC assay using calves blood showed better results. Several test substances were false negatives with rat blood. This high false negative rate would be correctly identified by the animal test but it may also lead to increased animal consumption. For that RBC assay with calf blood cells is preferable to the employment of rat blood as screening method with a reduction and refinement strategy.

Microfluidic sonicator for real-time disruption of eukaryotic cells and bacterial spores for DNA analysis

Biologic agent screening is a three-step process: lysis of host cell membranes or walls to release their DNA, polymerase chain reaction to amplify the genetic material and screening for distinguishing genetic signatures. Macrofluidic devices commonly use sonication as a lysis method. Here, we present a piezoelectric microfluidic minisonicator and test its performance. Eukaryotic human leukemia HL-60 cells and Bacillus subtilis bacterial spores were lysed as they passed through a microfluidic channel at 50 μL/min and 5 μL/min, respectively, in the absence of any chemical denaturants, enzymes or microparticles. We used fluorescence-activated cell sorting and hematocytometry to measure 80% lysis of HL-60 cells after 3 s of sonication. Real-time polymerase chain reaction indicated 50% lysis of B. subtilis spores with 30 s of sonication. Advantages of the minisonicator over macrofluidic implementations include a small sample volume (2.5 μL), reduced energy consumption and compatibility with other microfluidic blocks. These features make this device an attractive option for “lab-on-a-chip” and portable applications. (E-mail: theodore_marentis@hms.harvard.edu)

In vitro studies of the hemolytic activity of microemulsions in human erythrocytes

Hemolytic activity in human erythrocytes as alternative to in vivo testing was used as a potential screening method to evaluate irritant potential of microemulsions for possible application in pharmaceutical and cosmetic formulations. Microemulsions were prepared by mixing surfactants and oil and slowly titrating the mixtures with aliquots of phosphate buffer saline or water. All microemulsions were characterized by dynamic light scattering to determine both the mean droplet size and droplet distribution. Microemulsion droplet size decreased as aqueous component increased. No differences in droplet size were observed between formulations containing phosphate buffered saline or water. The hemolytic activity was measured photometrically by the RBC assay, based in the cell membrane lysis, to estimate the potential irritation of both surfactants and microemulsions selected with water or PBS as aqueous component. The most hemolytic microemulsions corresponded to those containing the surfactant Labrasol ®, with or without butyl lactate, and no differences were found between the hemolytic activity between these components and microemulsions containing them. The highest hemolytic activity of microemulsions in this study may be attributed to the excipient used in the formulations. We should avoid the use of high amounts of Labrasol ® and butyl lactate in microemulsions because they may be potential irritants.

Design and In-vitro Testing of Effective Poly( l-Lysine Iso-Phthalamide) Based Drug Targeting Systems for Solid Tumours

The mode of cell uptake, relative cytotoxicity and efficacy of poly ( l -lysine iso -phthalamide)-drug conjugates, PEG-graft poly ( l -lysine iso-phthalamide)-drug containing micelles, poly ( l -lysine iso-phthalamide) modified drug/protein nanospheres and unmodified protein nanospheres as potential delivery systems have been evaluated using doxorubicin and polymethine fluorophores as model drugs. pH dependent cell membrane lysis has been demonstrated using a haemolysis model and the cytotoxicity of the polymeric system evaluated using standard MTT and LDH activity assays. In-vitro toxicity and cellular targeting data are presented and the potential healthcare impact of such systems is discussed.

Questioning the Mechanism of Nerve Injury

We were struck by the title “Possible Mechanism of Irreversible Nerve Injury Caused by Local Anesthetics” that appeared recently in Anesthesiology.1Kitagawa et al. 1showed that “local anesthetics used clinically can form molecular aggregations at high concentrations, resulting in the appearance of detergent properties in these agents.” They concluded, “The mechanisms of irreversible neurologic injury induced by high concentrated local anesthetic seem likely to result from the detergent nature of local anesthetics.”In 1994, we published results of in vitro experiments regarding the irreversible conduction block associated with high concentrations of local anesthetics.2In that publication, we examined the changes in the compound resting potential (CRP) of the isolated frog sciatic nerve. The CRP, like the compound action potential (CAP), is an average of membrane potentials of all the fibers in the nerve bundle. The CRP became less negative (by 18 ± 2 mV, n = 3) when 5% lidocaine was placed in the drug exposure pool. The kinetics of this apparent depolarization consisted of a rapid phase of 5–10 mV in amplitude, occurring in less than 10 s, and a slow phase of 10–15 mV amplitude, taking 10–15 min to reach steady state. The CAP amplitude decreased to zero within 40 s of exposure to 5% lidocaine. On replacement of lidocaine by amphibian Ringer’s solution, this apparent depolarization reversed with a corresponding rapid and slow phase, and the CRP was restored to within 2–4 mV of its predrug value after a 2-h washout, although the CAP did not reappear. A similar depolarization of 24 ± 4 mV, consisting of rapid and slow phases, resulted when the nerve was exposed to 200 mm choline chloride dissolved in Ringer’s solution. In the choline chloride Ringer’s solution, however, the action potential amplitude only decreased by 58.8 ± 6.4% (n = 4) and recovered to within 3.2 ± 0.8% of the initial value after a 50-min wash in Ringer’s solution. It is likely that the rapid apparent depolarization is actually an ionic solution artifact resulting from the interaction of the silver–silver chloride electrode in the test pool with the increased [Cl−] present in both 5% (185 mm) lidocaine hydrochloride and 200 mm choline chloride. The mechanism of the slow phase is unclear, but both phases of this depolarization seem to result from differences in the ionic composition and not tonicity, because exposure of two nerves to Ringer’s solution containing 400 mm dextrose (7.2%, equally hypertonic to the lidocaine) changed the CRP by less than 0.4 mV, accompanied by small reductions of the CAP (11% and 18%) that remained after 2 h of washing in Ringer’s solution. In contrast, if the nerve was intentionally lysed by 5% sodium lauryl sulfate (an ionic detergent) in Ringer’s solution, the CRP disappeared within 3 min (a permanent depolarization of 30–40 mV),3accompanied by an irretrievable loss of the action potential.At first glance, the mechanism proposed by Kitagawa et al. seems plausible. However, our data suggest that lysis of the nerve cell membrane resulting from a detergent action is not the mechanism for irreversible nerve injury that we observed in an isolated nerve preparation. It seems more likely that the local anesthetic injury that we observed resulted from a “wrecking” of the sodium conductance system (lack of CAP generation) in the face of preserved membrane structure, ionic gradients, and CRP.* Boston University Medical School, Boston Medical Center, Boston, Massachusetts. donald.lambert@bmc.org

Detergent Effects of Sodium Deoxycholate Are a Major Feature of an Injectable Phosphatidylcholine Formulation Used for Localized Fat Dissolution

BACKGROUND Phosphatidylcholine injections are becoming an increasingly popular technique to treat localized fat accumulation. This formula is composed primarily of phosphatidylcholine and sodium deoxycholate, a bile salt used to solubilize the natural phospholipid in water. The mechanism through which this injectable phosphatidylcholine formulation causes localized fat reduction is unknown. OBJECTIVE To investigate the active component and mechanism of action of an injectable phosphatidylcholine formulation in clinical use. METHODS Cell viability and cell membrane lysis assays were performed on cell cultures and porcine skin after treatment with the phosphatidylcholine formula, isolated sodium deoxycholate, or common laboratory detergents Triton-X 100 and Empigen BB. In addition, we described the histologic changes after injection of these substances into porcine tissue. RESULTS A significant and comparable loss of cell viability, cell membrane lysis, and disruption of fat and muscle architecture was seen in cell cultures and tissue specimens treated with the phosphatidylcholine formula and isolated sodium deoxycholate. These findings were similar to the effects produced after treatment with laboratory detergents. CONCLUSIONS The phosphatidylcholine formula popularly used in subcutaneous injections for fat dissolution works primarily as a detergent causing nonspecific lysis of cell membranes. Our findings suggest that sodium deoxycholate is the major active component responsible for cell lysis. Detergent substances may have a role in eliminating unwanted adipose tissue. It is advised that physicians use caution until adequate safety data are available.

Detergent effects of sodium deoxycholate are a major feature of an injectable phosphatidylcholine formulation used for localized fat dissolution.

Phosphatidylcholine injections are becoming an increasingly popular technique to treat localized fat accumulation. This formula is composed primarily of phosphatidylcholine and sodium deoxycholate, a bile salt used to solubilize the natural phospholipid in water. The mechanism through which this injectable phosphatidylcholine formulation causes localized fat reduction is unknown. To investigate the active component and mechanism of action of an injectable phosphatidylcholine formulation in clinical use. Cell viability and cell membrane lysis assays were performed on cell cultures and porcine skin after treatment with the phosphatidylcholine formula, isolated sodium deoxycholate, or common laboratory detergents Triton-X 100 and Empigen BB. In addition, we described the histologic changes after injection of these substances into porcine tissue. A significant and comparable loss of cell viability, cell membrane lysis, and disruption of fat and muscle architecture was seen in cell cultures and tissue specimens treated with the phosphatidylcholine formula and isolated sodium deoxycholate. These findings were similar to the effects produced after treatment with laboratory detergents. The phosphatidylcholine formula popularly used in subcutaneous injections for fat dissolution works primarily as a detergent causing nonspecific lysis of cell membranes. Our findings suggest that sodium deoxycholate is the major active component responsible for cell lysis. Detergent substances may have a role in eliminating unwanted adipose tissue. It is advised that physicians use caution until adequate safety data are available.

The induction of eosinophil peroxidase release: improved methods of measurement and stimulation

The production and release of eosinophil peroxidase (EPO) has been associated with human pathology. Degranulation assays with eosinophils are typically very difficult to do, with very low release values. EPO is unique for its high cationic charge. As such, it adheres to most extracellular surfaces, rendering it more difficult to measure compared with other released cellular proteins. Based on the understanding of the sticky nature of EPO, we were concerned that EPO released in vitro cannot be reproducibly measured in the supernatants of stimulated cells. Instead, we suspected that much of the released EPO was left adherent to the tube walls. We chose to investigate the measurement of EPO activity using the peroxidase substrate, O-phenylenediamine (OPD). Unlike other peroxidase substrates, OPD is soluble in aqueous physiological solutions, which do not lyse cell membranes, thereby allowing us to add OPD directly to eosinophils and exclusively measure extracellular EPO. This novel approach would remove the concerns of incorrect EPO measurements due to its adhesive nature. In addition, we developed this method to quantify EPO release in terms of EPO concentration. Finally, using this technique, we have been able to demonstrate secretory IgA (s-IgA)-induced release of EPO. By using OPD, we have developed a more sensitive and specific method to analyze the release of extracellular EPO.

Interaction of electrically neutral and cationic forms of imipramine with liposome and erythrocyte membranes

Imipramine (IP) is an amphiphilic amine that is clinically useful as a tricyclic antidepressant. In the present work, we have investigated the pH effects on (a) the micellization properties of IP, (b) IP-induced release of aqueous contents from artificial vesicles (liposomes), and (c) IP-induced haemolysis, under hypotonic and isotonic conditions, in the pH range 5.0–10.0. Our data show that IP-membrane interaction caused important membrane destabilisation. However, changes in pH modified strongly the effects of IP. We suggest that the parameters of IP-induced model and cell membrane lysis are sensitive to the changes in the charge of this amphiphile. IP interacts more strongly with the membrane at basic pH (smaller net charge) than at neutral or acidic pH. A simple interpretation of these data is that increasing pH causes a decrease in IP net charge, thus a decrease in the critical micellar concentration of IP, and an increased partition into the lipid bilayer. Concomitantly, pH variations both above and below 7 tend to destabilise the cell membrane, so that the protective effect of IP against hypotonic haemolysis has a maximum at neutral pH.

An ultrastructural study on cytotoxic effects of mono(2-ethylhexyl) phthalate (MEHP) on testes in Shiba goat in vitro

In this study, the effects of mono(2-ethylhexyl) phthalate (MEHP), one of metabolites of di(2-ethylhexyl) phthalate, on immature Shiba goat testes in vitro were examined. The testes of 2-month-old Shiba goats were cut into smaller pieces, and seeded in medium. At 1, 3, 6 and 9 hr after administration of MEHP at various concentrations (0, 100 nmol ml(-1), 1 nmol ml(-1), and 1 x 10(-3) nmol ml(-1), respectively), the specimens were obtained for light and transmission electron microscopic observations. As a result, at 1 hr after exposure to MEHP, the vacuolization and nuclear membrane rupture appeared in Sertoli cells. Such alterations tended to gradually increase in number in time and dose-dependent manners. Moreover, by MEHP treatment, apoptotic spermatogenic cells (characterized with chromatin condensation, cytoplasm shrinkage without membrane rupture, still functioning cell organelles, and packed cell contents in membrane-bounded bodies), apoptotic Sertoli cells (characterized with nuclear membrane lysis, nuclear condensation), necrotic spermatogenic cells (characterized with swollen and ruptured mitochondria, plasma membrane lysis, spilt cell contents, and chromatin clumps), and necrotic Sertoli cells (characterized with marginated chromatins along the nuclear membrane, ruptured vesicles within the MNB, some swollen and ruptured cell organelles, e.g. mitochondria) could be identified. Conclusively, ultrastructurally the treatment with MEHP at low concentration tends to lead spermatogenic and Sertoli cells to apoptosis, whereas that at high concentration tends to lead spermatogenic and Sertoli cells to necrosis. Thus, the testicular tissue culture is advantageous for screening testicular toxicity of chemicals.

Effects of Mono(2-ethylhexyl) Phthalate (MEHP) on Testes in Rats In Vitro

The phthalate esters have been used as plasticizers for various plastic products, and their testicular toxicity has been reported. In this study, the effects of mono(2-ethylhexyl) phthalate (MEHP), one of the phthalate esters, on prepubertal rat testes in vitro were examined. The testes of 20-day-old Sprague Dawley (SD) rats were cut into smaller pieces and seeded in medium, and then the specimens were obtained for light and transmission electron microscopic observations. As a result, at 1 hr after exposure to MEHP, TUNEL-positive spermatogenic cells were identified, and they gradually increased in number in time- and dose-dependent manners. Ultrastructurally, apoptotic spermatogenic cells (characterized with chromatin condensation, cytoplasm shrinkage without membrane rupture, still-functioning cell organelles, and packed cell contents in membrane-bounded bodies), necrotic spermatogenic cells (characterized with swollen and ruptured mitochondria, plasma membrane lysis, spilt cell contents, and chromatin clumps), apoptotic Sertoli cells (highly condensed nuclei and nuclear membrane lysis) and necrotic Sertoli cells (marginated chromatins along the nuclear membrane, some swollen and ruptured cell organelles, e.g. mitochondria) could be identified. Conclusively, based on transmission electron microscopic observations, MEHP treatment may affect spermatogenic cells, and lead them to necrosis. Thus, testicular tissue cultures and cell cultures are of advantageous for screening testicular toxicity of chemicals.

Transmission electron microscopic observations of membrane effects of antibiotic cecropin B on Escherichia coli.

The pathway of cell membrane lysis by the peptide antibiotic cecropin B (CB), which contains both a hydrophobic and an amphipathic alpha-helix, was analysed by assessing the morphological changes of Escherichia coli following treatment with the peptide. Exposure of green fluorescent protein (GFP)-expressing E. coli to CB does not lead to an efflux of GFP. Moreover, transmission electron microscopic (TEM) examination of cecropin B-treated cells showed that severe swelling precedes cell death and that the outer membrane becomes distended away from the plasma membrane. Using immuno-gold staining and TEM of E. coli expressing the maltose-binding protein in the cytoplasm, it was apparent that the protein remains restricted to the cytoplasmic compartment. These observations suggest that CB causes gross disruption of the outer membrane of Gram-negative bacteria. Circular dichroism measurements of CB in the presence of cell membrane-mimicking liposomes showed that CB forms secondary structure dependent on the ratio of [lipid]/[peptide]. These observations from this study are important for the future design of custom antimicrobial peptides.