Primary cultured cells as sensitive in vitro model for assessment of toxicants-comparison to hepatocytes and gill epithelia

In an effort to develop cultured cell models for toxicity screening and environmental biomonitoring, we compared primary cultured gill epithelia and hepatocytes from freshwater tilapia (Oreochromis niloticus) to assess their sensitivity to AhR agonist toxicants. Epithelia were cultured on permeable supports (terephthalate membranes, "filters") and bathed on the apical with waterborne toxicants (pseudo in vivo asymmetrical culture conditions). Hepatocytes were cultured in multi-well plates and exposed to toxicants in culture medium. Cytochrome P4501A (measured as 7-Ethoxyresorufin-O-deethylase, EROD) was selected as a biomarker. For cultured gill epithelia, the integrity of the epithelia remained unchanged on exposure to model toxicants, such as 1,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), benzo(a)pyrene B[a]P, polychlorinated biphenyl (PCB) mixture (Aroclor 1254), and polybrominated diphenyl ether (PBDE) mixture (DE71). A good concentration-dependent response of EROD activity was clearly observed in both cultured gill epithelia and hepatocytes. The time-course response of EROD was measured as early as 3 h, and was maximal after 6 h of exposure to TCDD, B [alp and Aroclor 1254. The estimated 6 h EC50 for TCDD, B [a]P, and Aroclor 1254 was 1.2x10(-9), 5.7x10(-8) and 6.6x10(-6) M. For the cultured hepatocytes, time-course study showed that a significant induction of EROD took place at 18 h, and the maximal induction of EROD was observed at 24 h after exposure. The estimated 24 It EC50 for TCDD, B[a]P, and Aroclor 1254 was 1.4x10(-9), 8.1x10(-8) and 7.3x10(-6) M. There was no induction or inhibition of EROD in DE71 exposure to both gill epithelia and hepatocytes. The results show that cultured gill epithelia more rapidly induce EROD and are slightly more sensitive than cultured hepatocytes, and could be used as a rapid and sensitive tool for screening chemicals and monitoring environmental AhR agonist toxicants. (c) 2006 Elsevier B.V. All rights reserved.

Comparative studies on in vitro sperm decondensation and pronucleus formation in egg extracts between gynogenetic and bisexual fish

A cell-free system based upon the egg extracts from gynogenetic gibel carp (Carassius auratus gibelio) or bisexual red common carp (Cyprinus carpio red variety) was developed to investigate developmental behaviors of the demembranated sperm nuclei. Both red common carp and gibel carp sperm nuclei could decondense fully and form pronuclei in the red common carp egg extracts. Gibel carp sperm nuclei could also decondense fully and form pronuclei in the gibel carp egg extracts, but red common carp sperm nuclei could not decondense sufficiently in the same extracts. The significant differences of morphological changes were further confirmed by ultrastructural. observation of transmission electron microscopy. The data further offer cytological evidence for gonochoristic reproduction in the gynogenetically reproducing gibel carp. In addition, the sperm nuclei in vitro decondensation is dependent on the pH in the extracts, and the decondensed efficiency is optimal at pH 7. However, no DNA replication was observed in the two kinds of egg extracts during the incubation period of the sperm nuclei. It is suggested that the egg extracts prepared from the gynogenetic gibel carp should be a valid in vitro system for studying molecular mechanism on gynogenesis and reproduction mode diversity in fish.

Optimizing the formation of in vitro sponge primmorphs from the Chinese sponge Stylotella agminata (Ridley)

The establishment and optimization of in vitro primmorph formation from a Chinese sponge, Stylotella agminata (Ridley), collected from the South China Sea, were investigated. Our aims were to identify the key factors affecting primmorph formation in this species and to optimize the technique for developing an in vitro primmorph culture system. The size of dissociated cells from S. agminata is relatively small, in the range between 5 and 10 mum. Round-shaped primmorphs of less than 100 gm were formed 3 days after transferring the dissociated cells into seawater containing Ca2+ and Mg2+. The effect of various cell dissociation conditions, inoculum. cell density, concentration of antibiotics, pH, and temperature was further investigated upon the formation of primmorphs. The time required for primmorph formation, primmorph size distribution, and the proliferating capability were microscopically documented. Healthy sponge S. agminata, inoculum. cell density and culture temperature play a critical role for the successful formation of primmorphs and that the microbial contamination will have to be controlled. (C) 2002 Elsevier Science B.V. All rights reserved.

Feasibility of biodegradable PLGA common bile duct stents: An in vitro and in vivo study

The current study investigates the feasibility of using a biodegradable polymeric stent in common bile duct (CBD) repair and reconstruction. Here, poly(l-lactide-co-glycolide) (PLGA, molar ratio LA/GA = 80/20) was processed into a circular tube- and dumbbell-shaped specimens to determine the in vitro degradation behavior in bile. The morphology, weight loss, and molecular weight changes were then investigated in conjunction with evaluations of the mechanical properties of the specimen. Circular tube-shaped PLGA stents with X-ray opacity were subsequently used in common bile duct exploration (CBDE) and primary suturing in canine models. Next, X-ray images of CBD stents in vivo were compared and levels of serum liver enzymes and a histological analysis were conducted after stent transplantation. The results showed that the PLGA stents exhibited the required biomedical properties and spontaneously disappeared from CBDs in 4-5 weeks. The degradation period and function match the requirements in repair and reconstruction of CBDs to support the duct, guide bile drainage, and reduce T-tube-related complications.

Synthesized oversulphated, acetylated and benzoylated derivatives of fucoidan extracted from Laminaria japonica and their potential antioxidant activity in vitro

Three sulphated polysaccharide derivatives (oversulphated, acetylated and benzoylated fucoidan) were successfully synthesized, and their antioxidant activities were investigated employing various established in vitro systems. A novel catalyst N-bromosuccinimide (NBS) was used in acetylation and benzoylation reaction, and the degree of acetylation was evaluated using IR and NMR spectra. Both fucoidan derivatives possessed considerable antioxidant activity, and had stronger antioxidant ability than fucoidan in certain tests. The benzoylated fucoidan showed strongest superoxide and hydroxyl radical scavenging activity, however, the acetylated fucoidan exhibited strongest activity on scavenging DPPH radical and reducing power. Available data obtained with in vitro models suggested that substituted groups of fucoidan played an important role on antioxidant activity. The mechanism on the antioxidant activity of sulphonyl, acetyl and benzoyl group is different. (C) 2008 Elsevier Ltd. All rights reserved.

Antioxidant activity of differently regioselective chitosan sulfates in vitro

Differently regioselective chitosan sulfates were prepared according to Hanno Baumann's methods. Their antioxidant potencies were investigated employing various established in vitro systems, such as 1,1-diphenyl-2-picrylhydrazyl (DPPH)/superoxide/hydroxyl radicals scavenging, reducing power, iron ion chelating and total antioxidant activity. All kinds of sulfated chitosans (HCTS, TSCTS, SCTS, TCTS) showed strong inhibitory activity toward superoxide radical by the PMS-NADH system compared to Vc. According to the above-mentioned order their IC50 were 0.012, 0.040, 0.015, 0.022mg/mL, respectively, however, scavenging activity of Vc on superoxide radical was 68.19% at 2.0mg/mL. Scavenging activity of superoxide radical was found to be in the order of HCTS > SCTS > TCTS > TSCTS > Vc. Furthermore, all kinds of sulfated chitosans exhibited strong concentration-dependent inhibition of deoxyribose oxidation. Except for HCTS, others had stronger scavenging activity on hydroxyl radical than Vc. Scavenging effect of TSCTS on 1, 1 -diphenyl-2-picrylhydrazy] radical was little lower than that of BHA, but better than that of others. All kinds of sulfated chitosans were efficient in the reducing power, especially TSCTS. TSCTS and TCTS showed considerable ferrous ion chelating potency. The data obtained in vitro models clearly establish the antioxidant potency of all kinds of sulfated chitosans. These in vitro results suggested the possibility that sulfated chitosans could be effectively employed as ingredient in health or functional food, to alleviate oxidative stress. However, comprehensive studies need to be conducted to ascertain the in vivo safety of sulfated chitosans in experimental animal models. (C) 2004 Elsevier Ltd. All rights reserved.

Preparation of high-molecular weight and high-sulfate content chitosans and their potential antioxidant activity in vitro

The influence of molecular weight and substitution degree of sulfated polysaccharides on their biological activity is considered in majority of works involving the anticoagulant or antiviral properties of these substances. Therefore, the present paper describes the effect of preparation conditions of sulfated chitosans on their molecular weight and sulfur content, such as different reaction time, acid solvent and temperature. Foregoing literature expounded the action of dichloroacetic acid (DCAA) as acid solvent in homogeneous reaction. However, DCAA is expensive and noxious, therefore, in the present paper cheap and non-noxious formic acid (88%) was in place of DCAA. Furthermore, during reaction formic acid was not dehydrated. Under formic acid we obtained the satisfying results that was higher yield and equivalent sulfur contents compared to DCAA. IR and C-13 NMR spectrums proved the structure of the resultant obtained under formic acid or DCAA to be same. Now, it has not been reported for formic acid as acid solvent in homogeneous reaction of chitosan sulfatation. In this present paper, we also determined antioxidant activity of high-molecular weight and high-sulfate-content chitosans (HCTS). The results showed that HCTS could scavenge superoxide and hydroxyl radical. Its IC50 is 0.012 and 3.269 mg/mL, respectively. It had obviously reducing power and slight chelating activity. The data obtained in in vitro models clearly establish the antioxidant potency of HCTS. It is a potential antioxidant in vitro. (C) 2005 Elsevier Ltd. All rights reserved.