Intra-laboratory validated human cell-based in vitro vasculogenesis/angiogenesis test with serum-free medium.

Vasculogenesis and angiogenesis are the processes by which new blood vessels are formed. We have developed a serum-free human adipose stromal cell and umbilical cord vein endothelial cell based vasculogenesis/angiogenesis test. In this study, the test was validated in our GLP laboratory following the OECD Guidance Document 34 [1] using erlotinib, acetylic salicylic acid, levamisole, 2-methoxyestradiol, anti-VEGF, methimazole, and D-mannitol to show its reproducibility, repeatability, and predictivity for humans. The results were obtained from immunostained tubule structures and cytotoxicity assessment. The performance of the test was evaluated using 26 suspected teratogens and non-teratogens. The positive predictive value was 71.4% and the negative predictive value was 50.0%, indicating that inhibition of vasculogenesis is a significant mechanism behind teratogenesis. In conclusion, this test has great potential to be a screening test for prioritization purposes of chemicals and to be a test in a battery to predict developmental hazards in a regulatory context.

Evaluation of the cytotoxicity of selected systemic and intravitreally dosed drugs in the cultures of human retinal pigment epithelial cell line and of pig primary retinal pigment epithelial cells.

The cytotoxicity of the selected systemic and intravitreally dosed drugs tamoxifen, toremifene, chloroquine, 5-fluorouracil, gentamicin and ganciclovir was studied in retinal pigment epithelium (RPE) in vitro. The cytotoxicity was assayed in the human RPE cell line D407 and the pig RPE cell culture using the WST-1 test, which is an assay of cell proliferation and viability. The effects of experimental conditions on the WST-1 test (cell density, serum content in the culture medium, the exposure time) were evaluated. The EC50 values in tamoxifen-treated D407 cells ranged between 6.7 and 8.9 micromol/l, and in pig RPE cells between 10.1 and 12.2 micromol/l, depending on the cell density used. The corresponding values for toremifene were 7.4 to 11.1 micromol/l in D407 cells and 10.0 to 11.6 micromol/l in pig RPE cells. In chloroquine-treated cells, the EC50 values were 110.0 micromol/l for D407 cells and 58.4 micromol/l for pig RPE cells. Gentamicin and ganciclovir did not show any toxicity in micromolar concentrations. The exposure time was a significant factor, especially when the drug did not induce cell death, but was antiproliferative (5-fluorouracil). Serum protected the cells from the toxic effects of the drugs. Both cell cultures were most sensitive to tamoxifen and toremifene, and next to chloroquine. The drug toxicities obtained in the present study were quite similar in both cell types; that is, the pig RPE cells and the human D 407 cell line, despite the differences in, for example, the growth rate and melanin contents of the cell types. Owing to the homeostatic functions important for the whole neuroretina, RPE is an interesting in vitro model for the evaluation of retinal toxicity, but, in addition to the WST-1 test, more specific tests and markers based on the homeostatic functions of the RPE are needed.

Effects of mercuric chloride exposure on the glutamate uptake by cultured retinal pigment epithelial cells.

The cytotoxicity of mercuric chloride and the effects of mercuric chloride on glutamate and calcium uptake and the factors regulating glutamate uptake were studied in retinal pigment epithelium (RPE) cell cultures. RPE cells isolated from pig eyes and human RPE cell line (D407) cells were cultured to confluency and further subcultured according to the test protocol in question. The cytotoxicity caused by 15 min of exposure to mercuric chloride (0.01--1000 microM) was evaluated by WST-1 assay based on the activity of mitochondrial dehydrogenases. [(3)H]Glutamate uptake was measured after the cells were exposed to 0.1--100 microM mercuric chloride and the selected regulators of protein kinase C (PKC) pathway: PKC activator SC10, PKC inhibitor chelerythrine chloride, phospholipase A(2)/C inhibitor manoalide, tyrosine kinase inhibitor lavendustin A, competitive NMDA receptor antagonist AP7 and IP(3) receptor antagonist heparin. Intracellular calcium was monitored with Fluo-3 probe starting immediately after the exposure to 1--1000 microM mercuric chloride. Mercuric chloride showed concentration-dependent effects on cell viability, on glutamate uptake and on intracellular calcium concentration. The results give some support to the concept that glutamate uptake is affected by PKC. The PKC inhibitor chelerythrine chloride decreased glutamate uptake by 25%, but the PKC activator SC10 could partly prevent the inhibitory effect of mercuric chloride. Lavendustin A, manoalide and heparin had smaller, but statistically significant, effects. All these substances act on mediators which can regulate the activity of PKC. However, PKC is not likely to be the only regulator of glutamate uptake. The rise observed in [Ca(2+)](i) may initiate various cellular events during mercury intoxication.

The phagocytosis of rod outer segments is inhibited by selected drugs in retinal pigment epithelial cell cultures.

The effects of tamoxifen, toremifene and chloroquine on the phagocytosis of rod outer segments by retinal pigment epithelium were evaluated in human retinal pigment epithelial cell line D407 and pig retinal pigment epithelial cell culture. Retinal pigment epithelial cells were exposed to different concentrations of tamoxifen (1-20 microM), toremifene (1-20 microM) and chloroquine (1-1000 microM), and challenged with FITC-labeled rod outer segments for 24 hr. The phagocytized (bound and ingested) rod outer segments were measured fluorometrically, and the effect of the drugs on the phagocytosis was determined. The cytotoxicity of the drugs was evaluated by measuring their effects on mitochondrial enzyme activities (WST-1-test). The results showed that the test compounds inhibited the phagocytosis of rod outer segments in both D407 and pig retinal pigment epithelial cells. The phagocytic activity was more sensitive to tamoxifen (EC(50) 7.2 microM for D407 cells and 3.6 microM for pig retinal pigment epithelial cells) and toremifene (EC(50) 6.2 microM and 3.1 microM respectively) than to chloroquine (EC(50) 77.2 microM for D407 cells). The inhibition of rod outer segment phagocytosis in both cell cultures started at lower dose levels of test compounds than the cytotoxicity indicated by the WST-1-test. The experiments were carried out both in serum-free medium and serum-containing medium. Serum seemed to be a critical factor in the medium and caused difficulties in the interpretation of the results.

Piperonyl butoxide potentiates the synaptosome ATPase inhibiting effect of pyrethrin.

Pyrethrins are widely used insecticides in both agriculture and households. In many commercial formulations piperonyl butoxide (PBO) is used with pyrethrins. PBO is a well-known synergist of pyrethrins, used to intensify their effects. One of the cellular targets of pyrethrins is the sodium channel in the membrane. In the present study, the activity of the membrane-bound integral protein ATPase was studied as a biomarker for the membrane effects of pyrethrin and PBO. Cerebral synaptosomes of rat brain were used in the study. The isolation of synaptosomes was performed with the Percoll gradient method. Both total ATPase and Mg2+ activated ATPase were studied by determining inorganic phosphate. Exposure to 0.1-1000 microM of pyrethrin and to 0.4-4000 microM of PBO decreased ATPase activity dose-dependently. The most efficient mixture was the one consisting of one part of pyrethrin and four parts of PBO. The activity of total ATPase decreased 15% in concentrations of 0.1-10 microM pyrethrin, and a 50% decrease was found at 100 microM pyrethrin. The mixture of pyrethrin and PBO caused a 15-60% decrease in the total ATPase activity at 0.1-10 microM pyrethrin and 0.4-40 microM PBO. A 85% decrease was found after exposure to the mixture of 100 microM pyrethrin and 400 microM PBO. PBO alone had no effect at 0.4-40 microM concentrations, but a marked effect was seen at over 40 microM concentrations. The results indicate that PBO is an effective synergist of pyrethrin and that it is very toxic in high concentrations. The results also confirm that neuronal sodium homeostasis is one target of the neurotoxic effect of pyrethroid compounds.

Retinal müller cell culture.

A mini-review is presented of the current techniques for maintaining Müller cells in a culture. Within the retina, Müller cells are the predominant glial cells. These highly specialised cells extend over the entire neural retina. One of the most important of the various physiological functions of Müller cells is to regulate the balance of ions and neurotransmitters in the retina. Disturbance of these regulatory functions may lead to toxic effects on receptor and other neural cells in the neuroretina, and may be a common mechanism of clinical retinal neuropathy. The main excitatory neurotransmitter in the retina is glutamate. Müller cells regulate the amount of glutamate in the synaptic regions of the neural network in the retina. Accumulation of extra glutamate seems to be an important mechanism for initiating pathological changes leading to retinal damage. Many previous in vitro studies on the role of Müller cells in retinal toxicology have been based on the use of morphological and histochemical methods. In cell toxicology studies, it is important to develop culture techniques able to provide more cells for biochemical determinations.

Retinal pigment epithelial cell cultures as a tool for evaluating retinal toxicity in vitro.

This article reviews in vitro testing of retinal toxicity in retinal pigment epithelium (RPE) cell cultures. It is based on the literature on RPE cell cultures and on our recent studies on the retinal toxicity of selected amphiphilic drugs. The RPE plays a major role in maintaining the homeostasis and health of the retina. Various pharmacological agents are known to cause adverse effects in RPE cells. For example, long-term treatment with chloroquine in patients with rheumatoid arthritis has induced retinopathy, and tamoxifen, a drug that is commonly used in the treatment of advanced breast cancer and in the prevention of breast cancer among high-risk women, has been reported to cause retinal changes and impaired vision. During our research, we have developed novel in vitro methods for evaluating the retinal toxicity of xenobiotics. We have used a pig RPE primary culture and a human RPE cell line (D407), which retain epithelial cell characteristics. They form a layer of hexagonal cells with intercellular junctions, and possess a keratin-containing cytoskeleton. They are both good models for determining the retinal cell toxicity of test compounds. Further studies on phagocytic activity, lysosomal enzyme activity and glutamate uptake might generate new methods for the toxicological evaluation of the retinal side-effects of drugs in vitro.

Effects of tamoxifen, toremifene and chloroquine on the lysosomal enzymes in cultured retinal pigment epithelial cells.

Retinal pigment epithelial cells carry out phagocytosis and digestion of material shed from the photoreceptor outer segments. In this process, the integrity of lysosomal enzymes is of major importance. In the present study the effects of tamoxifen, toremifene and chloroquine on the activity of two lysosomal enzymes (cathepsin D and N-acetyl-beta-D-glucosaminidase) in the retinal pigment epithelial cells were studied. Retinal pigment epithelial cells from pig eyes were cultured for two weeks in Dulbecco's Modified Eagle Medium, after which the cells were exposed to 1-40 microM concentrations of tamoxifen citrate, toremifene citrate and chloroquine diphosphate. To eliminate possible medium-borne oestrogenic mechanisms, the test was repeated using phenol red-free medium with charcoal-stripped fetal calf serum. The exposure time was one week, after which the lysosomal enzymes cathepsin D and N-acetyl-beta-glucosaminidase were determined. Cellular injuries were assessed by quantifying the leakage of lactate dehydrogenase into the culture medium. Cathepsin D and N-acetyl-beta-D-glucosaminidase showed different sensitivities to tamoxifen, toremifene and chloroquine. The main lysosomal protease cathepsin D was more sensitive than N-acetyl-beta-D-glucosaminidase to the effects of tamoxifen and toremifene, possibly due to their antioestrogenic properties. The phenol red-free medium with charcoal-stripped serum seemed to make the drugs more effective than the reference medium. Chloroquine had only a minor effect on the lysosomal protease cathepsin D, but a clearer effect could be seen on N-acetyl-beta-glucosaminidase.

Retinal pigment epithelium cell culture as a model for evaluation of the toxicity of tamoxifen and chloroquine.

The retinal pigment epithelium (RPE) removes the outer segments of photoreceptor cells by phagocytosis. We studied the effects of tamoxifen and chloroquine on the activity of the lysosomal enzymes N-acetyl-beta-glucosaminidase and cathepsin D in RPE in vitro to evaluate the possible eye toxicity caused by these drugs. The results show decreases in the activities of lysosomal enzymes after drug exposure. The enzymes tested seemed to be more sensitive to tamoxifen than to chloroquine. A profound decrease in the activities of the lysosomal enzymes only started at concentrations above therapeutic dose levels.

Effects of mercury, methylmercury and aluminium on glial fibrillary acidic protein expression in rat cerebellar astrocyte cultures.

The effects of mercuric chloride, methylmercury chloride and aluminium chloride on glial fibrillary acidic protein (GFAP) expression in primary cerebellar astrocyte cultures were studied. GFAP has been found to be a quantitative marker of neuronal injuries on the central nervous system in vivo. The GFAP content of the astrocytes was examined by sandwich ELISA after exposure for 1 wk to various metal concentrations in the culture medium. Cellular injuries were assessed by quantifying the lactate dehydrogenase (LDH) leakage into the culture medium. The expression of GFAP was found at lower dose levels than the leakage of LDH, indicating that the expression of GFAP could be a more sensitive marker of neurotoxicity than LDH leakage. In morphological examination, staining with monoclonal antibody showed GFAP induction after mercury exposure.

Molecular cloning and nucleotide sequence of chicken avidin-related genes 1-5.

Using avidin cDNA as a hybridisation probe, we detected a gene family whose putative products are related to the chicken egg-white avidin. Two overlapping genomic clones were found to contain five genes (avidin-related genes 1-5, avr1-avr5), which have been cloned, characterized and sequenced. All of the genes have a four-exon structure with an overall identity with the avidin cDNA of 88-92%. The genes appear to have no pseudogenic features and, in fact, two of these genes have been shown to be transcribed. The putative proteins share a sequence identity of 68-78% with avidin. The amino acid residues responsible for the biotin-binding activity of avidin and the bacterial biotin-binding protein, streptavidin, are highly conserved. Since avidin is induced in both a progesterone-specific manner and in connection with inflammation, these genes offer a valuable tool to study complex gene regulation in vivo.