Induction of pulmonary fibrosis in organ-cultured rat lung by cadmium chloride and transforming growth factor-beta1.

Cadmium chloride (CdCl2) exposure has been reported to induce pulmonary fibrosis in rats. Accumulating evidence has shown that cytokines play a pivotal role in the excessive production of connective tissue components in pulmonary fibrosis. In this report, rat lung slice cultures were used to study the synergistic involvement of transforming growth factor-beta1 (TGF-beta1) in CdCl2-induced alveolar fibrosis. Rat lung slices were maintained at the interphase of air and medium on a polyester mesh stretched on a plastic scaffold. Treatment of lung slices with 2.5, 5 or 10 microM CdCl2 for 7 days resulted in 85, 40 and 6% respectively for relative survival. Under these culture conditions, CdCl2 alone did not induce alveolar fibrosis in rat lung slices. However, in the presence of 0.5 ng/ml TGF-beta1, CdCl2 at a dose ranging from 1 to 5 microM increased the thickness of alveolar septa. Furthermore, the thickness of alveolar septa in lung slices treated with CdCl2 was dose-dependently increased by the presence of TGF-beta1. The thickened alveolar septa were apparently due to the deposition of excessive extracellular matrix, as revealed by trichrome stain and ultrastructural examination. Our results also show that fibrogenic activity induced by the combined treatment with CdCl2 and TGF-beta1 can be reduced by co-treatment with 200 microg/ml lambda-carrageenan, a TGF-beta1 inhibitor. Therefore, the present results indicate that TGF-beta1 can synergistically stimulate the fibrogenic activity in lung tissue subsequent to CdCl2 injury.

Reactive oxygen species are involved in nickel inhibition of DNA repair.

Nickel has been shown to inhibit DNA repair in a way that may play a role in its toxicity. Since nickel treatment increases cellular reactive oxygen species (ROS), we have investigated the involvement of ROS in nickel inhibition of DNA repair. Inhibition of glutathione synthesis or catalase activity increased the enhancing effect of nickel on the cytotoxicity of ultraviolet (UV) light. Inhibition of catalase and glutathione peroxidase activities also enhanced the retardation effect of nickel on the rejoining of DNA strand breaks accumulated by hydroxyurea plus cytosine-beta-D-arabinofuranoside in UV-irradiated cells. Since DNA polymerization and ligation are involved in the DNA-break rejoining, we have investigated the effect of ROS on these two steps in an extract of Chinese hamster ovary cells. Nickel inhibition of the incorporation of (3H)dTTP into the DNase I-activated calf thymus DNA was stronger than the ligation of poly(dA) x oligo(dT), whereas H2O2 was more potent in inhibiting DNA ligation than DNA polymerization. Nickel, in the presence of H2O2, exhibited a synergistic inhibition on both DNA polymerization and ligation and caused protein fragmentation. In addition, glutathione could completely recover the inhibition by nickel or H2O2 alone but only partially recover the inhibition by nickel plus H2O2. Therefore, nickel may bind to DNA-repair enzymes and generate oxygen-free radicals to cause protein degradation in situ. This irreversible damage to the proteins involved in DNA repair, replication, recombination, and transcription could be important for the toxic effects of nickel.

Magnesium reduces nickel inhibition of DNA polymerization.

The activities of DNA polymerization and DNA ligation in extract of Chinese hamster ovary cells were both stimulated by MgCl2. DNA polymerization was stimulated by MgCl2 above 0.25 mM, whereas, MgCl2 above 2 mM was required to stimulate DNA ligation. The activity of DNA polymerization maintained a plateau at MgCl2 1-12 mM, whereas DNA ligation reached a maximal activity at MgCl2 6 mM and decreased thereafter. NiCl2 0.1-0.2 mM also had a stimulatory effect on DNA polymerization, but was much less potent than MgCl2. However, nickel ion (Ni2+) had no detectable stimulating effect on the activity of DNA ligation. In the presence of MgCl2, the activities of DNA polymerization and DNA ligation decreased with increasing concentration of NiCl2. Ni2+ inhibition of DNA polymerization was reduced by increasing the concentration of MgCl2, but increasing the concentration of MgCl2 did not reduce Ni2+ inhibition of DNA ligation. Preincubating cell extract with MgCl2 decreased the Ni2+ inhibition of DNA polymerization but not DNA ligation. These results suggest that Ni2+ may compete with magnesium ion (Mg2+) to reduce DNA polymerization, but this mechanism seems not applicable to Ni2+ inhibition of DNA ligation.

Differential cytotoxicity of cadmium to rat embryonic fibroblasts and human skin fibroblasts.

In order to identify why cadmium is differentially toxic to humans and rats, we compared cadmium-induced cytotoxic responses in rat embryonic fibroblasts (REF) and human skin fibroblasts (HFW). According to the values of the lethal concentration 50% for cadmium, REF were about 13-fold more sensitive than HFW to cadmium acetate (1.5 vs 25 microM). Furthermore, progression of S phase cells was more severely delayed by cadmium in REF than in HFW. At doses that killed 90% of REF or HFW (5 or 50 microM, respectively), 50% of DNA synthesis was inhibited in REF, whereas DNA synthesis was not apparently inhibited in HFW. The differential sensitivity to cadmium could not be simply due to different basal levels of metallothionein, since the cellular metallothionein content in HFW was only 1.6 times that in REF, and metallothionein was apparently induced by cadmium in both cells with similar synthesis rates. Furthermore, elevation of cellular metallothionein levels by zinc sulfate pretreatment decreased cadmium toxicity in both cell types, but did not alter their relative sensitivity to cadmium. The differential sensitivity was also not due to differences in cadmium accumulation, since HFW accumulated more cadmium than REF after a 24-hr exposure to 1 or 5 microM cadmium acetate. Although most of the cadmium remained in the cytoplasm, the nuclei of REF contained 12-fold more cadmium than nuclei of HFW (1.31 vs 0.11 micrograms Cd/mg nuclear protein). Therefore, our results indicate that a high level of cadmium accumulation in nuclei of REF may be responsible for cell killing through breakdown of nuclear functions.

Glutathione can rescue the inhibitory effects of nickel on DNA ligation and repair synthesis.

The purpose of this investigation was to explore the reason why nickel chloride enhances the cytotoxicity and genotoxicity of ultraviolet (UV) light, but not that of methyl methanesulfonate (MMS) in Chinese hamster ovary cells. The cellular glutathione content was increased by treatment with MMS or nickel, but not with UV. Post-treatment with nickel synergistically raised the cellular glutathione content in MMS-treated cells; this phenomenon was not observed in UV-irradiated cells. Preventing cellular glutathione induction by buthionine sulfoximine increased the cytotoxicity, the frequency of sister chromatid exchange and prolonged the cell cycle in cells treated with nickel or MMS plus nickel. Pretreatment with N-acetylcysteine, a glutathione precursor, increased the clonogenic survival of cells treated with UV plus nickel. In vitro assays indicated that nickel could inhibit oligonucleotide ligation and the repair synthesis of UV- or MMS-treated plasmids and glutathione could relieve nickel inhibition. These results suggest that the enhancement by nickel of UV cytotoxicity and genotoxicity may be due to its inhibition of DNA repair, whereas treating cells with MMS plus nickel increased cellular glutathione levels, which may help in neutralizing the toxicity of nickel. The results also suggest that the activity of gamma-glutamylcysteine synthetase, the rate-limiting enzyme in glutathione biosynthesis, may be increased by treatment with MMS, nickel and more so with MMS plus nickel.

Characterization of a heat-resistant strain of Tilapia ovary cells.

Tilapia ovary cells (TO-2) cease to proliferate when moved from normal growth temperature of 31 degrees C to 37 degrees C, and arrest in G1 and G2 phases of the cell cycle. The ability of the arrested cells to re-enter the cell cycle when restored to 31 degrees C decreases inversely with time spent at 37 degrees C. A heat-resistant strain, TO-37c, cloned from the surviving fraction of TO-2 after heat treatment, has been found to re-enter the cell cycle with greater facility and to have a higher rate of survival. TO-37c cells have a smaller cell volume than TO-2 and show a distinct morphology at 37 degrees C. Most of the heat-shock proteins (hsps) induced on temperature change were similar, but in TO-37c the decline in the synthesis of a 27 X 10(3) Mr hsp was faster and a 37 degrees C-specific 60 X 10(3) Mr hsp was missing. Ultraviolet (u.v.) sensitivity was slightly affected if heat treatment was given after irradiation. However, when cells were preheated and then u.v. irradiated, the u.v. sensitivity increased sharply for TO-2 cells but not for TO-37c.

Thermal adaptation and heat shock response of Tilapia ovary cells.

The growth of tissue culture TO-2 cells derived from the warm water fish Tilapia, the induction of thermotolerance, and protein synthesis profiles of these cells in response to temperature changes were examined. TO-2 cells can grow between 15 to 34 degrees, with an optimal growth temperature of 31 degrees. There is no apparent killing of the cells when the temperature is lowered to 4 degrees for up to 3 days. Survival of TO-2 cells at 43 degrees was studied after various preheat treatments: 1) acute heating at 40 degrees for 15 min followed by 31 degrees incubation, 2) chronic exposure at 37 degrees for several hr, or 3) long-term thermal adaptation at 34 degrees. The cells acquire thermotolerance from pre-exposure to 37 degrees for as short as 6 hr. Preheating at 40 degrees followed by incubation at 31 degrees also induces thermotolerance against a subsequent 43 degrees heat challenge. In addition, 34 degrees thermal adapted cells are resistant to 43 degrees heating. One- and two-dimensional gel electrophoresis of proteins after heat treatments show that three major heat shock proteins with molecular weights around 87, 70, and 27 kD are preferentially synthesized. The synthesis of two additional proteins with an isoelectric point of 6.9 and molecular weights of 60 and 44 kD are significantly enhanced in 34 degrees thermal-adapted and 37 degrees chronic heated cells, but not in cells subjected to an acute heat shock at either 40 degrees or 43 degrees. On the other hand, the 27 kD heat shock proteins are mainly present in the 43 degrees, 40 degrees, and 37 degrees heat-shocked cells, but not in the 34 degrees thermal-adapted cells.

DNA replication and repair of Tilapia cells. II. Effects of temperature on DNA replication and ultraviolet repair in Tilapia ovary cells.

TO-2 is a fish cell line derived from the Tilapia ovary. It grows over a wide range of temperature (15-34 degrees C). While most fish cells lack DNA excision repair and are hypersensitive to ultraviolet light (u.v.), Tilapia cells are more u.v.-resistant than mammalian cells. In this paper we report the effects of temperature on DNA replication and u.v. repair in TO-2 cells. When the cells were moved from 31 degrees C to the sublethal high temperature of 37 degrees C, the rate of DNA synthesis first decreased to 60%, then speedy recovery soon set in, and after 8 h at 37 degrees C the rate of DNA synthesis overshot the 31 degrees C control level by 180%. When moved to low temperature (18 degrees C) Tilapia cells also showed an initial suppression of DNA synthesis before settling at 30% of the control level. u.v. reduced but could not block DNA synthesis completely. The inhibition was overcome in 3 h at 37, 31 and 25 degrees C, but not at 18 degrees C. Initiation of nascent DNA synthesis was blocked at 4 J m-2 in TO-2 cells compared with less than or equal to 1 J m-2 in mammalian cells. After 9 J m-2 u.v. irradiation, low molecular weight DNA replication intermediates started to accumulate, and they could be chased into high molecular weight DNA with little delay. TO-2 cells showed low levels of u.v.-induced excision repair; but this was prominent compared with other fish cells. The u.v.-induced incision rate has been measured at various temperatures, and the activation energy of incision estimated to be 13 kcal mol-1 (1 cal approximately equal to 4.184 J).

DNA replication and repair in Tilapia cells. I. The effect of ultraviolet radiation.

The effect of ultraviolet radiation on a cell line established from the warm water fish Tilapia has been assessed by measuring the rate of DNA synthesis, excision repair, post-replication repair and cell survival. The cells tolerate ultraviolet radiation better than mammalian cells with respect to DNA synthesis, post-replication repair and cell survival. They are also efficient in excision repair, which in other fish cell lines has been found to be at a low level or absent. Their response to the inhibitors hydroxyurea and 1-beta-D-arabinofuranosylcytosine is less sensitive than that of other cell lines, yet the cells seem to have very small pools of DNA precursor.

Ultraviolet-induced DNA excision repair in human B and T lymphocytes. III. Repair in lymphocytes from chronic lymphocytic leukaemia.

There have been conflicting reports about the capacity of lymphocytes from individuals with chronic lymphocytic leukaemia (CLL) to undertake u.v.-induced DNA repair. We have examined repair in CLL cells and in control and age-matched purified B and T lymphocytes by a technique independent of incorporation of radioactive precursor, i.e. by the recovery of normal sedimentation behaviour of nucleoid bodies obtained from these cells by lysis in high salt and non-ionic detergent. Recovery of normal sedimentation is associated with restoration of DNA supercoiling. CLL cells were found to be as sensitive to u.v. and to repair at similar rates as age-matched B controls. They are considerably more sensitive than young B cells and repair less efficiently. Reasons for the reported discrepancies in CLL repair are discussed.