In vitro Study on Role of Hsp70 Expression in DNA Damage of Human Embryonic Lung Cells Exposed to Benzo[a]pyrene

Objective Benzo[a]pyrene (B[a]P), a ubiquitous environmental pollutant, is a potent procarcinogen and mutagen that can elicit tumors, leading to malignancy. Heat shock proteins (Hsp) have been shown to protect cells against damages caused by various stresses including exposure to numerous chemicals. Whether Hsps, or more specifically Hsp70, are involved in repair of B[a]P-induced DNA damage is currently unknown. Methods We assessed the potential role of the inducible form of Hsp70 in B[a]P-induced DNA damage of human embryonic lung (HEL) cells using immunoblot and the comet assay (i.e., the single cell gel electrophoresis assay). Results Exposure to B[a]P induced a dose-dependent decrease in the level of Hsp70, but a dose-dependent +-increase in DNA damage both in untreated (control) HEL cells and in cells preconditioned by a heat treatment. Heat preconditioning prior to B[a]P exposure potentiated the effect of B[a]P at a low dose (10 μmol/L), but appeared to be protective at higher doses. There was a negative correlation between Hsp70 level and DNA damage in the non-preheated as well as in the preconditioned cells. Conclusion These data suggest that exposure of HEL cells to B[a]P may induce a dose-dependent reduction in the levels of the inducible Hsp70. The detailed mechanisms for the reduction of Hsp70 levels by B[a]P and the role of Hsp70 in DNA damage under different concentrations of B[a]P remains to be determined.

Genotoxic and Nongenotoxic Effects of Glycidyl Methacrylate on Human Lung Fibroblast Cells

Objective To evaluate the genotoxic and nongenotoxic effects of short-term exposure to glycidyl mathacrylate (GMA) on human lung fibroblast cells (2BS cells) in vitro. Methods DNA strand breakage was determined by single cell gel electrophoresis, and DNA ladder formation assay and flow cytometric analysis were carried out to detect apoptic responses of cells to GMA exposure. The HPRT gene mutation assay was used to evaluate the mutagenicity, and the effect of GMA on gap junctional intercellular communication (GJIC) in the exposed cells was examined with the scrape loading/dye transfer technique. The ability of GMA to transform 2BS cells was also tested by an in vitro cell transformation assay. Results Exposure to GMA resulted in a dose-dependent increase in DNA strand breaks but not apoptic responses. GMA was also shown to significantly induce HPRT gene mutations and morphological transformation in 2BS cells in vitro. In contrast, GMA produced a concentration-dependent inhibition of GJIC. Conclusions GMA elicits both genotoxic and nongenotoxic effects on 2BS cells in vitro. The induction of DNA damage and gene mutations and inhibition of GJIC by GMA may casually contribute to GMA-induced cell transformation.

A Study on Cellular Reactions and Fibrogenic Effectsof Mineral Dusts

In vivo cytotoxicity including cellular metabolic activity, lysozyme content and total protein content in rat bronchoalveolar lavage, capacity of interleukin-1 released from rat pulmonary cells and fibrogenic effects evaluated from rat lung dry weight, collagen content of the whole lung and pathological grading induced by mineral dust were assayed. The results showed that: (1) The relationship among in vivo cytotoxicity, interleukin-1 release,fibrogenic effects on the lung induced by mineral dusts correlated well with the free SiO2content in mineral dusts in most (but not all) cases; (2) The biological harmful effects of mixed dusts were not simply the additive effect of single dust. In the group of WO3-SiO2mixture, the fibrogenicity was mainly due to SiO2, tungsten trioxide (WO3) showed neither fibrogenic effect, nor significant potentiality to enhance SiO2 fibrogenicity, while in the group of SnO2-SiO2, SnO2 was suppressive to the effect of SiO2, although the contents of SiO2 in the two mixed dusts were similar

Studies on Biochemical Mechanism of Neurotoxicity Induced by Acrylamide in Rats

The effects of acrylamide on calmodulin (CaM), cAMP, cGMP, Ca2+, Mg2+-ATPase and 45Ca2+ uptake in nervous system were determined in Wistar rats (ip, 10 or 50 mg-kg-1 for 12 d). The results indicate that acrylamide caused an alteration in calcium homeostasis in rat brain by decreasing the Ca2+-sequestering capacity of microsomes, and this may occur due to an efflux of calcium secondary to a microsomal structure damage. The changes of CaM in nervous system coupled with potential alteration in the intracellular Ca2+ concentration could affect many CaM-dependent enzymes (i.e. Ca2+, Mg2+-ATPase) and cyclic AMP system, and CaM or cAMP is known to be 'lexicological second messenger' that could initiate neurotoxicity, though more work is needed to elucidate the details of the mechanism.

Determination of Acrylamide Metabolite,Mercapturic Acld by High Performance Liquid Chromatography

A HPLC Assay was developed to identify and measure the metabolite of acrylamide, mercapturic acid, N-Acetyl-s-(propionamide)-cysteine (APC) in urine. O-phthalaldehyde (OPA) was utilized as a precolumn derivatizing agent in the assay. This derivative was isolated with a good selectivity by high performance liquid chromatography (HPLC) employing reversed phase ODS columns. The quantitation of the mercapturic acid derivative was reproducible and with a detection limit of 1 pmol. The average coefficient of variation for the runs carried out on the same day was approximately 4.6% at the range of 80-160 ianol-L'1 of APC, and the mean analytical recovery from urine samples was 94%.The metabolite of urine of workers exposed to acrylamide was identified as N-acetyl-s-(propionamide)- cysteine by HPLC chromatography and fluorescence scan and HPLC-Mass spectra. All results were identical with the authentic synthesized compound.

Determination of Pyrethroids in Human Urine by Gas Chromatography

A gas chromatographic method for the determination of deltamethrin and fenvalerate in human urine is described. Both deltamethrin and fenvalerate have been analysed by gas chromatography with a electron capture detector. The least detectable concentration of both pyrethroids in urine is 0.2 g per litre. The precision and accuracy of the method were within acceptable limits. The method has been used to determine the pyrethroids in urine samples obtained from packers, spraymen and acute pyrethroids poisoning patients

Studies on Structural Changes of Collagen in Silicosis

In order to provide scientific information on the prevention and treatment of silicosis,studies about changes of silicotic collagen in lungs were carried out. In this paper, we present experiments about the structural changes of collagen in silicotic lungs of rats and patients. These included electron microscopy, circular dichroism and infrared spectroscopy studies of collagen fibers. The results indicated that fibers of silicotic collagen were shorter in length, smaller in diameter and decreased in α-helix content. The -Si-O-R- group and -OH group were found increased and -C-C- backbone shortened. The increase of -Si-O-R-group indicated that silica formed linking bridges between collagens which may be the cause of progressive enlargement of nodules

STUDY ON GMA-DNA ADDUCTS

Objective. DNA modification fixed as mutations in the cells may be an essential factor in the initiation step of chemical carcinogenesis. In order to explore the mechanism of gene mutation and cell transformation induced by glycidyl methacrylate (GMA), the current test studied the characteristics of GMA DNA adducts formation in vitro. Methods. In vitro test, dAMP, dCMP, dGMP, dTMP and calf thymus DNA were allowed to react with GMA (Glycidyl Methacrylate). After the reaction, the mixtures were detected by UV and subjected to reversed phase HPLC on ultrasphere ODS reversed phase column, the reaction products were eluted with a linear gradients of methanol (solvent A) and 10mmol/L ammonium formate, pH5 0 (solvent B). The synthesized adducts were then characterized by UV spectroscopy in acid (pH1 0), neutral (pH7 2), alkaline (pH11 0) and by mass spectroscopy. Results. The results showed that GMA could bind with dAMP, dCMP, dGMP and calf thymus DNA by covalent bond, and the binding sites were specific (N 6 of adenine, N 3 of cytosine). Meanwhile, a main GMA DNA adduct in the reaction of GMA with calf thymus DNA was confirmed as N 3 methacrylate 2 hydroxypropy1 dCMP. Conclusions. GMA can react with DNA and /or deoxynucleotide monophosphate and generate some adducts such as N 6 methacrylate 2 hydroxypropyl dAMP and N 3 methacrylate 2 hydroxypropyl dCMP, ets. Formation of GMA DNA adducts is an important molecular event in gene mutation and cell transformation induced by GMA.

Spectrum of Glycidyl Methacrylate-induced Mutation in Plasmid-Escherichia coli System

In order to chitracterizc the speclrum of mutallon indueed by glyeidyl methacrylule(GMA ), the plasmid pBR322 was modificed with this mutagen in vitro , transttclcd intoapproprate Escherichia colii host HB101. The mutants were then sereened and defined by DNA sequencing . Sequence analysis reveals that G MA induces two classes of mulations deletion of the mono-. di' or fetra-base or the insertion of mono' or di-base. Both typesof mutations, with about 10 % frequency. occur predominantly at C-G runs and at5'-CNCCN-3' sequence ,which are hotspots for GMA damage and may cause frameshift mutation.

MOLECULAR MUTAGENESIS INDUCED BY GLYCIDYL METHACRYLATE

Glycidyl methacrylate (GMA)is a recently recognized mutagen. In order to explore the mutagenicity and mechanism of GMA, plasmid PBR322 was used for in vitro binding, mutant screening, restriction enzyme mapping,and DNA sequencing. To explore the mechamism by which an initial premutational event is converted into a stable heritable mutation, pBR322 and GMA-bound pBR322 were transformed into E. coli HB101 , and the following results were obtained : 1) GMA-bound PBR322 induced phenotype changes in competent cells. Two stable and heritable mutants were isolated (Ap￣RTc￣S and Ap￣STc￣R). 2) When restriction enzyme mapping was used to analyze the mutant Ap￣RTc￣S , four of seven maps showed changes, but no large DNA insertion or deletion were observed.3) The frequency of deletion and insertion forms counted about 10%. Sequence specificity and hot spot regions were evident in the sequence analysis of mutated plasmid. The above results indicate that the premutagenic lesions of plasmid induced by GMA can be converted into point mutations in vivo.

MOUSE ANTIBODY RESPONSE FOLLOWING REPETITIVE INJECTIONS OF GAMMA－IRRADIATED HUMAN PLACENTA COLLAGEN

Injectable bovine collagen has been used clinically for years. But both the necessity of repeated injections to maintain corrections and the question of adverse allergic reactions developing from the use of a xenogenic collagen have been an area of serious concern. To overoome these adverse effects, we have developed injectable collagen preparations from human placenta. Gamma irradiation was used for sterilization and crosslinkins of the collagen.We observed the mouse immune respose to gamma-irradiated human placenta soluble and insoluble collagen following multiple injections. After six injections of these materials, no total IgG level increase was found, nor was antibody specifically directed against human collagen found.Mouse antibody levels were also observed following Zyderm II and Zyplast repetitive injections and following repetitive implantations of coated vicryl and chromic gut. No humoral immune response was found in this heterologous type system.

Potential Use of Luminol-Dependent Chemiluminescence for Estimation of Free Radicals Produced in Hepatic Microsomes and Reconstituted Cytochrome P-450 Systems

The evaluation of luminol-dependent chemiluminescence (CL) for determination of free radicals generated during the oxidative reactions of microsomal enzymes in microsomes and reconstituted systems has been studied. The results indicated that the CL not only depended on NADPH induction but also on intact cytochrome P-450 enzymes. The light emission signal was strongly scavenged by SOD with inhibition rate of 93 %. A concentration of 24 /μmol.L-1 cadmium acetate and 2.5 μmol.L-1 mercuric chloride also inhibited the signal significantly as compared with the control. The study of CL in detecting free radicals formed by biotransformation of 44 xenobiotics suggested that some of the compounds known as free radicals producing agents by bioactivation increased the light emission in cytochrome P-450 enzyme system, such as carbon tetrachlonde, chloroform, tetrachloroethylene etc., the others forming toxic metabolites by biotransformation induced the CL in some cases, such as carbon disulfide, benzene, methyldursban, methylparathion etc. or decreased the light emission, such as parathion, aniline and polychlorinated byphenyls etc., and still others whose toxic effects have no relationship with their biotransformation had no influence or an inhibitory effect on the CL, such as aminopyrine, TOCP, and acrylamide. CL in the cytochrome P-450 enzyme system remained unchanged during the biotransformation of carcinogens such as diethylnitrosamine, dipropymitrosamine and benzo (a) pyrene as compared with the control. However, 3-methylcholanthrene and dimethyInitrosamine had an enhancing effect on the CL, while 2-acetylaminofluorene and 2-aminoanthracene showed an inhibition to the signals.

Relation of IL-2,IL-3 and IL-4 with Allergic Asthma Induced by Spores of Mushroom(Pleurotus sapidus)

The effects of IL-2, IL-3, and IL-4 activities on occurrence of allergic asthma induced by spores of mushroom have been studied by measuring the IL-2, IL-3, and IL-4 activities in culture supernatants of spleen lymphocytes from guinea pigs being stimulated with Con A. The IL-4 activity (14.9±0.18 u) in the culture supernatant was higher than control group (6.7±1.5 u). The IL-2 and IL-3 activities were all similar to that of the control group. The IL-4 synthesis was similarly raised as the IL-4 activity. These results indicated that there is a relationship between the occurrence of allergic asthma and the increase of IL-4.