In vitro effects of triclosan and methyl-triclosan on the marine gastropod Haliotis tuberculata

Triclosan (2,4,4'-trichloro-2'-hydroxy-diphenyl ether; TCS) is an antibacterial agent incorporated in a wide variety of household and personal care products. Because of its partial elimination in sewage treatment plants, TCS is commonly detected in natural waters and sediments. Moreover, due to its high hydrophobicity, TCS accumulates in fatty tissues in various aquatic organisms. TCS can be converted into methyl-triclosan (2,4,4'-trichloro-2'-methoxydiphenyl ether; MTCS) after biological methylation. In this study, the acute cytotoxicity of TCS and MTCS in short-term in vitro experiments was assessed on cell cultures from the European abalone Haliotis tuberculata. The results showed that morphology and density of hemocyte are affected from a concentration of 8 μM TCS. Using the XTT reduction assay, TCS has been demonstrated to decrease hemocyte metabolism activity in a dose- and time-dependent exposure. The IC(50) was evaluated at 6 μM for both hemocyte and gill cells after a 24 h-incubation with TCS. A significant cytotoxicity of MTCS was also observed from 4 μM in 24 h-old hemocyte culture. Our results reveal a toxic effect of TCS and MTCS on immune (hemocytes) and/or respiratory cells (gill cells) of the abalone, species living in coastal waters areas and exposed to anthropogenic pollution.     

Effects of a mixture of growth factors and proteins on the development of the osteogenic phenotype in human alveolar bone cell cultures.

Strategies to promote bone repair have included exposure of cells to growth factor (GF) preparations from blood that generally include proteins as part of a complex mixture. This study aimed to evaluate the effects of such a mixture on different parameters of the development of the osteogenic phenotype in vitro. Osteoblastic cells were obtained by enzymatic digestion of human alveolar bone and cultured under standard osteogenic conditions until subconfluence. They were subcultured on Thermanox coverslips up to 14 days. Treated cultures were exposed during the first 7 days to osteogenic medium supplemented with a GFs + proteins mixture containing the major components found in platelet extracts [platelet-derived growth factor-BB, transforming growth factor (TGF)-beta1, TGF-beta2, albumin, fibronectin, and thrombospondin] and to osteogenic medium alone thereafter. Control cultures were exposed only to the osteogenic medium. Treated cultures exhibited a significantly higher number of adherent cells from day 4 onward and of cycling cells at days 1 and 4, weak alkaline phosphatase (ALP) labeling, and significantly decreased levels of ALP activity and mRNA expression. At day 14, no Alizarin red-stained nodular areas were detected in cultures treated with GFs + proteins. Results were confirmed in the rat calvaria-derived osteogenic cell culture model. The addition of bone morphogenetic protein 7 or growth and differentiation factor 5 to treated cultures upregulated Runx2 and ALP mRNA expression, but surprisingly, ALP activity was not restored. These results showed that a mixture of GFs + proteins affects the development of the osteogenic phenotype both in human and rat cultures, leading to an increase in the number of cells, but expressed a less differentiated state.     

Influence of cryoprotective agent and cooling rate on frozen and thawed hemocytes from the Mollusk Haliotis tuberculata

Cultures of circulating cells from abalone (Haliotis tuberculata) may be used in fundamental research or in biotechnology. This paper describes attempts to develop a cryoconservation method for these hemocytes in order to constitute a standardized cell stock. Among a panel of five distinct cryoprotective solutions, 10% v/v glycerol ('G solution') was the most effective and better post-thaw recovery was achieved after cooling at 1 degrees C/min than after more rapid cooling (3 degrees C or 9 degrees C/min). In 2-day-old cultures, cell viability, assessed by DNA or protein content, was 83 and 78%, respectively, and metabolic activity, measured by the MTT reduction assay, reached 96%. Viability rates were only slightly reduced after 6 days of culture, suggesting a low proportion of damaged cells among the surviving hemocytes. This study identified a cryoprotective solution and a freezing protocol that allow thawed hemocytes to recover a large part of their viability.     

Cryopreservation of mantle dissociated cells from Haliotis tuberculata (Gastropoda) and postthawed primary cell cultures

Dissociated mantle cells from the gastropod mollusc Haliotis tuberculata were cultured after a freezing-thawing procedure using either 10% dimethyl sulfoxide (Me(2)SO) or 10% glycerol (Gly) as a cryoprotector. The survival rate of 2-day-old cultured cryopreserved cells after thawing, based on analysis of DNA and protein contents, was nearly 80% in comparison with 2-day-old cultured fresh cells. Cells thawed after cryopreservation exhibited the maintenance of all tested physiological activities. Metabolic activity (measured by the MTT test) and the activity of alkaline phosphatase (a plasma membrane-bound enzyme) were not decreased in comparison to those in cultured fresh cells. In addition, cryopreserved cultured cells maintained a physiological stimulation ability in response to treatment with growth factors. These results taken together represent one of the most convincing demonstrations of the survival and of the recovery of intact functional activities of molluscan cells after a freeze-thawing procedure. Our results suggest that in the future primary cultures of cryopreserved mantle cells will be able to be used for fundamental research, in toxicity tests, or in the field of biotechnology.     

Effects of establishing cell cultures and cell culture conditions on the proliferative life span of human fibroblasts isolated from different tissues and donors of different ages

It is well known that normal human cells placed in a culture environment exhibit a limited proliferative capacity. The extent to which the culture environment influences proliferative life span is not understood. This study evaluated the effects of the standard procedures used to establish and maintain cultures on the proliferative life spans of different types of human fibroblast cells established from fetal and adult skin and lung. The results of this study demonstrate that procedures to establish cell cultures use only one of several subpopulations of cells present in biopsy pieces and that the culture conditions routinely employed by most laboratories can exert significant effects on proliferative life-span determinations. The maximum proliferative life span differed significantly when obtained by growing the cells in two commonly used commercial media. Proliferative life span was inversely related to ambient oxygen tension and directly related to seeding density in all of the lines examined although lines established from adult skin were much more resistant to toxicity. Enzymatic antioxidant defense levels of fetal skin fibroblasts were much lower than those observed in adult skin fibroblasts, but the effects of oxygen on their life spans were similar. Hyperoxia induced larger increases in glutathione concentration in cell lines with low antioxidant enzyme levels.     

Comparative studies on the nutrition of two species of abalone,Haliotis tuberculata Linnaeus and Haliotis discus hannai Ino I. Effects of algal diets on growth and biochemical composition

Summary

This study was conducted to examine the nutritional value of eight algal diets for two species of abalone, Haliotis tuberculata and Haliotis discus hannai, by measuring biochemical composition of the algae and relating this to feeding rate, growth and biochemical composition of the animals. Nutritional value of algal diets can be divided into three categories for each species of abalone. For H. tuberculata the best performance was on the mixed diet and Palmaria palmata intermediate was Alaria esculenta, Ulva lactuca and Laminaria digitata, and lowest growth was on Laminaria saccharina and Chondrus crispus. For H. discus hannai, best performance was on A. esculenta, P. palmata and the mixed diet; intermediate was on L. saccharina and L. digitata and lowest was on U. lactuca. It is generally accepted that high “balanced” levels of protein (>15%), lipid (3–5%) and carbohydrate (20–30%), with no detrimental substances in natural algae are essential for optimal growth performance of these abalone. The fact that A. esculenta, L. saccharina and U. lactuca had different dietary values for the two abalone species indicates specific nutritive requirements and/or digestive physiology. Overall, H. tuberculata grew faster, had higher food conversion efficiencies and muscle yield than H. discus hannai. Generally abalone fed on the highest category diets, had higher muscle yields and levels of protein, visceral lipids and muscle carbohydrate. Viscera and foot muscle are reservoirs for lipid and carbohydrate, respectively. The effect of algal diet on sexual maturation is similar to that on somatic growth.     

Effects of vertebrate growth factors on digestive gland cells from the mollusc Pecten maximus L.: an in vitro study

In relation with the digestive cycle, the digestive gland cells of bivalve molluscs undergo a sequence of cytological changes which is controlled by external and internal effectors such as putative gastrointestinal hormones and growth differentiation factors. A tissue dissociation method was developed to investigate the in vitro effect of the vertebrate growth and differentiation factors: insulin, insulin growth factor I (IGF-I), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) on the digestive gland cells of the scallop Pecten maximus. All these vertebrate peptides induced a dose-dependent increased incorporation of ³H-leucine and ¹⁴C-uridine in whole digestive gland cell suspensions. However, after Percoll density gradient purification of the digestive cells, only stem and undifferentiated enriched cell fractions were responsive to the different peptides. In addition, insulin and IGF-I, but not EGF and bFGF, stimulated ³H-leucine incorporation in control dispersed mantle edge cells. These results suggest that insulin-related peptides could work as general growth promoting factors in molluscs. On the other hand, EGF and bFGF, or at least their molluscan counterparts, may be efficient growth differentiation factors in the regenerative processes occurring in the digestive gland of molluscs. Accepted: 26 September 1997     

Effects of different sterols on the inhibition of cell culture growth caused by the growth retardant tetcyclacis

Cell division in cell suspension cultures can be completely blocked by the growth retardant tetcyclacis at a concentration of 10^-4 mol l^-1. In rice cells it has been demonstrated that the growth inhibition can be completely overcome by application of cholesterol independent of the duration of pretreatment with tetcyclacis. In suspension cultures of maize and soybean, too, the effect of tetcyclacis on cell division was neutralized by adding cholesterol. Other plant sterols, stigmasterol, campesterol and sitosterol were active in a decreasing order. Modifications in the cholesterol perhydro-cyclopentanophenanthrene-ring system indicate that the hydroxyl group at C-3 and the double bond between C-5 and C-6 in ring B are required for the activity. In contrast, gibberellic acid as well as ent-kaurenoic acid could not compensate retardant effects. Likewise, tetcyclasis did not change the level of gibberellins in rice cells as shown by radioimmunoassay. Thus, it is concluded that in cell suspension cultures sterols play a more important role in cell division than gibberellins.Abbreviation GA_x gibberelin A_x     

Development and utilization of crustacean long-term primary cell cultures: Ecdysteroid effects in vitro

Summary

Long-term maintenance of lobster, Homarus americanus and crayfish, Pacifasticus leniusculus primary cell cultures of testicular and hematopoietic tissues, for 11 and 3 months, respectively, succeeded in a modified Medium 199 supplemented with 10% fetal bovine serum (pH 7.5, 200°C). In addition, NaC1 was used to adjust the lobster culture medium to 1000 mOsm and the crayfish medium to 400 mOsm. Proline concentration was also elevated. Testes were dissociated with 200 U/ml type II collagenase 2–3 days prior to culture.

Lobster hemocytes reacted to 10^?7 M 20-hydroxyecdysone (20-HE) by reducing contact inhibition and increasing invasive behavior one week after hormonal exposure. The presence of 10^?7 M 20-HE caused mesodermal cell death and spermatogonial proliferation in lobster testicular cell cultures within one week. Crayfish testicular mesodermal cells formed vacuoles 5 days after exposure to 10^?8 M 20-HE. These results are discussed in relation to the cellular events that occur in vivo during premolt.     

Effect of mitogenic factors extracted from fetal lung fibroblasts on the in vitro growth of melanocytes obtained from normal and vitiligo subjects

The effects of growth factors extracted from a newly established fetal lung fibroblast cell line (PMR-GF) on the melanocytes cultured from the Perilesional and dePigmented skins of vitiligo subjects and from normal healthy donors have been investigated. Melanocytes from normal subjects grown in the Presence of 10 ng/ml of 12-0-tetradecanoyl Phorbol l3-acetate and 10^-11 M cholera toxin grew exPonentially immediately after seeding the ePidermal cell susPensions. Exogenous addition of PMR-GF to these cells enhanced their growth rates. The Perilesional skin melanocytes of vitiligo subjects in most cases did not manifest any growth when cultured in the Presence of 12-0-tetradecanoyl Phorbol l3-acetate and cholera toxin. PMR-GF induced a brief burst of growth in these cells after a lag of 15 days. Vitiligo lesions gave rise to a few unPigmented dendritic cells that did not manifest any growth in the Presence or absence of PMR-GF. MorPhologically the Perilesional skin melanocytes of most vitiligo subjects, when cultured in 12-0-tetradecanoyl Phorbol l3-acetate and cholera toxin, aPPeared to be larger and hyPer-melanotic as comPared to those of normal individuals. In the Presence of PMR-GF these melanocytes aPPeared to be normal in size and less hyPermelanotiC. Our results indicate that the melanocytes from vitiligo subjects are defective and thus the basic defect in vitiligo could be with the melanocytes themselves.     

幼虫密度对草地螟血细胞数量和组成的影响

为了阐明幼虫密度对草地螟Loxostege sticticalis L.（鳞翅目： 螟蛾科）细胞免疫能力的影响, 本研究调查了在活体灰菜植株上1,5,10和20头/瓶（900 mL）4种密度条件下的其5龄幼虫血细胞种类、数量和组成。结果表明： 草地螟幼虫血淋巴中有原血细胞、浆血细胞、 颗粒血细胞、珠血细胞和类绛色血细胞等5种（类）血细胞。血细胞总数、 浆血细胞、颗粒血细胞数量随幼虫密度的增加而显著递增, 但原血细胞、珠血细胞和类绛色血细胞数量在幼虫密度间的差异不明显;各种血细胞所占血细胞总数的比例在4个密度中的排序相同, 但10和20头/瓶密度下的浆血细胞比例显著高于1头/瓶的,其余4种血细胞的比例在不同密度之间无显著差异。可见, 幼虫密度主要是通过影响草地螟幼虫浆血细胞和颗粒血细胞的数量及血细胞总数, 从而影响草地螟的细胞免疫能力。     

Effects of plating density and age in culture on growth and cell division of neonatal rat heart primary cultures

Summary The cell-type composition of the initial cell population from protease-dispersed neonatal rat heart tissue has been evaluated using time lapse photography and identification of cell type-specific functions. The effects of two commonly employed plating densities on growth and cell division of the two major cell types were examined. Total protein synthesis rates were not affected by plating density but did change with age in culture. Maximum protein synthesis rates were observed during the period of maximum cell division and cell growth (increase in total cell protein), which was from 24 h in culture to the 4th d in culture. After 6 d in culture, synthesis rates for total proteins remained constant for at least 2 wk. Sizing of cells by Coulter counter analysis indicated that essentially all the cells were increasing in size with age in culture. Measurements of cell numbers and rate of DNA synthesis indicated that the extent of cell division was dependent on plating density. Cells disaggregated from neonatal rat hearts consisted of approximately 75% muslce cells and 25% nonmuscle cells. This composition approximates the cell-type composition of the intact neonatal rat heart. In high density cultures there is little cell division and the relative proportionsof the cell types are preserved with time in culture. In low density cultures, proliferation of nonmuscle cells is a significant process and the composition of the cell population changes drastically during the first 2 to 3 d in culture. These results suggest that the low plating density used by many researchers may limit correlation of data derived from such cultures with the physiological state. It also indicates that plating densities should be given in published accounts for comparisons to be made with results from other laboratories. This work was supported in part by U.S. Public Health Service Grant HL10018 and The Pennsylvania State University Agricultural Experiment Station and was authorized for publication as Paper 5490 in the journal series of the Pennsylvania Agricultural Experiment Station.     

Milk-derived growth factors as serum supplements for the growth of fibroblast and epithelial cells

Summary We have investigated the response of several epithelial and fibroblastic cells to a mitogenic extract of bovine milk. Cation exchange chromatography was used to produce a mitogen-rich fraction from an industrial whey source that, although comprising only 0.5% of total whey protein, contained the bulk of the growth factor activity. This fraction was a source of potent growth promoting activity for all mesodermal-derived cells tested, including human skin and embryonic lung fibroblasts, Balb/c 3T3 fibroblasts, and rat L6 myoblasts. Maximal growth of all these cell types exceeded that observed in 10% fetal bovine serum. Feline kidney and baby hamster fibroblasts and Chinese hamster ovary cells were less responsive, achieving a maximal growth response of 50–75% that observed in 10% fetal bovine serum. Maximal growth achieved in whey-extract-supplemented cultures of Balb/c 3T3 and human skin fibroblasts, and L6 myoblast cultures exceeded that seen in response to recombinant acidic or basic fibroblast growth factor, platelet-derived growth factor, insulin-like growth factor, or epidermal growth factor. Importantly, addition of low concentrations of fetal bovine serum to the whey-derived mitogenic fraction produced an additive response. However, concentrated milk-derived factors were found to be inhibitory to the growth of all epithelial lines tested, including rat intestinal epithelial cells, canine kidney epithelial cells, and mink lung cells. It is concluded that industrial whey extracted in this form constitutes an important source of potent growth-promoting agents for the supplementation of mesodermal-derived cell cultures.     

Effects of tributyltin and benzo[a]pyrene on the immune-associated activities of hemocytes and recovery responses in the gastropod abalone, Haliotis diversicolor

Our previous study reports that short-term exposure to sublethal concentrations of benzo[a]pyrene (BaP) induces immunomodulation in the gastropod abalone, Haliotis diversicolor. In the present study, it was further observed that long-term chronic exposure to sublethal concentrations of BaP modulated the immunocompetence of abalones in terms of the change in activity of the antioxidant and immune associated parameters tested. In addition, the effect of tributyltin (TBT), another important genotoxicant in the aquatic environment, was investigated. Exposure of abalones to sublethal concentrations of TBT and BaP for 21 days resulted in significant decrease of total hemocyte count, phagocytosis, membrane stability and lysozyme activity. Conversely induction of extra and intra cellular superoxide generation, nitric oxide, nitric oxide synthase and myeloperoxidase activity was present when the abalones were exposed to TBT and BaP. Most of the immune associated parameters tested showed clear time dependent response to both toxicants. Within 14 days after the 21 day exposure to BaP, recovery was observed as evidenced by most of the parameters returning to their normal level. However, no recovery was observed within 14 days after the 21 day exposure to TBT as evidenced by continued elevation of intra cellular superoxide and nitrite production and decrease in THC, membrane stability and lysozyme activity. This suggested a prolonged TBT-induced impact on the immune reaction and possibly more damage than that caused by BaP. Overall the results suggest that chronic exposure to sublethal concentrations of TBT or BaP causes modulations in the immunocompetence of abalones with most of the immune associated parameters tested being stimulated, and this might be harmful to the host.     

Identification of extractable growth factors from small intestinal submucosa

When implanted as a biomaterial for tissue replacement, selected submucosal layers of porcine small intestine induce site-specific tissue remodeling. Small intestinal submucosa (SIS), as isolated, is primarily an acellular extracellular matrix material. In an attempt to discover the components of small intestinal submucosa which are able to induce this tissue remodeling, the material was extracted and extracts were tested for the ability to stimulate Swiss 3T3 fibroblasts to synthesize DNA and proliferate. Each of the four different extracts of small intestinal submucosa had measurable cell-stimulating activity when analyzed in both a whole cell proliferation assay (alamarBlue dye reduction) and a DNA synthesis assay ([³H]-thymidine incorporation). Proteins extracted from SIS with 2 M urea induced activity profiles in the two assays which were very similar to the activity profiles of basic fibroblast growth factor (FGF-2) in the assays. As well, the changes in cell morphology in response to the extracted proteins mimicked the changes induced by FGF-2. Neutralization experiments with specific antibodies to this growth factor confirmed the presence of FGF-2 and indicated that it was responsible for 60% of the fibroblast-stimulating activity of the urea extract of small intestinal submucosa. Western blot analysis with a monoclonal antibody specific for FGF-2 detected a reactive doublet at approximately 19 kDa and further confirmed the presence of FGF-2. Cell stimulating activity of proteins extracted from SIS with 4 M guanidine was neutralized by an antibody specific for transforming growth factor β (TGFβ). Changes in the morphology of the fibroblasts exposed to this extract were nearly identical to changes induced by TGFβ. Although no reactive protein band was detected at 25 kDa in nonreduced western blot analysis, several bands were reactive at higher molecular weight. The identity of this TGFβ-related component of small intestinal submucosa is unknown. Identification of FGF-2 and TGFβ-related activities in SIS, two growth factors known to significantly affect critical processes of tissue development and differentiation, provides the opportunity to further elucidate the mechanisms by which this extracellular matrix biomaterial modulates wound healing and tissue remodeling. J. Cell. Biochem. 67:478–491, 1997. © 1997 Wiley-Liss, Inc.     

Effects of exogenous interleukin-1β on primary sporocysts of Schistosoma mansoni (Trematoda) incubated with plasma and hemocytes from schistosome-susceptible and resistant Biomphalaria glabrata (Gastropoda)

Abstract. The cytokine interleukin-1β (IL-1β) mediates interactions of immune and inflammatory cells in mammals. Previous reports also have linked plasma (cell-free hemolymph) levels of IL-1β in the snail Biomphalaria glabrata to resistance against Schistosoma mansoni . In the present study, fluorescent probes were used to study larval schistosome and snail hemocyte viability during in vitro encounters. Hemolymph (plasma and hemocytes) from schistosome-susceptible (M-line) and resistant (13–16-R1) B. glabrata was added to sporocysts of S. mansoni and the viability of hemocytes and parasites was assessed. Next, IL-1β was added to sporocyst-hemolymph samples, the viability of sporocysts and hemocytes determined and then compared to control assays. The number of live sporocysts present after incubation for 1 h with hemolymph from M-line snails was significantly greater than the number seen when hemolymph from 13–16-R1 snails was tested. Nearly all sporocysts survived the 1 h incubation with M-line hemolymph, and most of the hemocytes attached to sporocysts were dead. In contrast, nearly all sporocysts were dead when hemolymph from 13–16-R1 snails was tested, and most attached hemocytes were alive. Addition of IL-1β to M-line hemolymph resulted in a dramatic increase in sporocyst death. Addition of IL-1β to 13–16-R1 hemolymph produced a small but significant increase in the rate of sporocyst death. These results indicate that the concentration of IL-1β present in hemolymph from B. glabrata is directly related to the ability of this snail to kill S. mansoni sporocysts in vitro.     

Effects of environmental factors on survival, growth, and production of American shad larvae

Episodic increases in temperature of 5°C above 20° C, over 48 h or declines in pH of 1·0 unit from pH 7·0 reduced survival of yolk-sac and feeding-stage larvae of American shad Alosa sapidissima . Over 16 days all measures of survival, growth, and production were more favourable at each higher temperature in the 15–25° C range. More favourable responses were also obtained at the higher prey level (500 v . 50 Artemia nauplii l^-1) and at the higher pH (7·5 v . 6·5). Combinations of high temperature and high prey levels, at pH 7·5, led to highest larval production. Little growth or production occurred at 15° C, regardless of pH or prey level. The effect of pH was strong with respect to survival, but weak with respect to growth. In attempts to restore American shad populations by larval stocking, release times and sites can be critical to optimize survival and eventual returns. Releases of larvae potentially will be most effective when made at temperatures >20° C, pH>7·0, and prey levels >50 1^-1. These conditions are most likely to occur in Maryland tributaries of Chesapeake Bay between mid-May and early June.     

The usefulness of CHAPS as a non-cytotoxic stabilizing agent in purification of growth factors

Among several detergents, a zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), was found to be least cytotoxic for cultured mammalian cells. CHAPS improved the activity recovery and elution profile of crude and purified fibroblast growth factors (FGFs) during chromatographies. Diluted preparations of FGFs were stabilized by CHAPS against the loss during storage. Amino acid sequence analysis was not disturbed by CHAPS. CHAPS was removable by reversed-phase high-performance liquid chromatography. These results indicate that CHAPS is useful as a non-cytotoxic stabilizing agent in purification of various kinds of bioactive polypeptides.Abbreviations -MEM Alpha Modification of Eagle's Minimal essential medium - CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate - CHAPSO 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane sulfonate - CS Calf Serum - EGF Epidermal Growth Factor - FGF Fibroblast Growth Factor - HPLC High-Performance Liquid Chromatography - NGF Nerve Growth Factor - NOG 1-O-n-octyl--D-glucopyranoside - NP-40 Nonidet P-40 - PBS Phosphate-Buffered Saline - SB 12 3-(dodecylmethylammonio)-1-propane sulfonate - SDS Sodium Dodecyl Sulfate - TGF- and Transforming Growth Factor type and