Cytotoxic, genotoxic, and mutagenic effects of diphenyl diselenide in Chinese hamster lung fibroblasts

Diphenyl diselenide (DPDS) is an electrophilic reagent used in the synthesis of a variety of pharmacologically active organic selenium compounds, and may increase the risk of human exposure to this chemical at the workplace. In a previous study, we demonstrated the pro-oxidant action and the mutagenic properties of this compound on bacteria and yeast. In the present study, we evaluated the putative cytotoxic, pro-oxidant, genotoxic, and mutagenic properties of this molecule in V79 Chinese lung fibroblast cells. When cells were treated with increasing concentrations of DPDS, its cytotoxic activity, as determined using four cell viability endpoints, occurs in doses up to 50 microM. The MTT reduction was stimulated, which may indicate reactive oxygen species (ROS) generation. Accordingly, the treatment of cells for 3h with cytotoxic doses of DPDS increased TBARS levels, and sensitized cells to oxidative challenge, indicating a pro-oxidant effect. The measurement of total, reduced, and oxidized glutathione showed that DPDS can lead to lower intracellular glutathione depletion, with no increase in the oxidation rate in a dose- and time-dependent manner. At the higher doses, DPDS generates DNA strand breaks, as observed using the comet assay. The treatment also induced an increase in the number of binucleated cells in the micronucleus test, showing mutagenic risk by this molecule at high concentrations. Finally, pre-incubation with N-acetylcysteine, which restored GSH to normal levels, annulled DPDS pro-oxidant and genotoxic effects. These findings show that DPDS-induced oxidative stress and toxicity are closely related to intracellular level of reduced glutathione. Moreover, at lower doses, this molecule has antioxidant properties, protecting the cell against oxidative damage induced by hydrogen peroxide.     

Cytotoxic and mutagenic effects of tobacco-borne free fatty acids

Tobacco smoke contains substances capable of binding iron in an aqueous medium and transferring the metal into both organic solvents and intact mammalian red cells. This iron-binding activity is due to free fatty acids which are abundant in tobacco smoke and form 2:1 (free fatty acid:iron) chelates with ferrous iron. These earlier observations suggested that smoke-borne free fatty acids and the associated delocalization of iron within the lung might contribute to both the chronic pulmonary inflammation and the carcinogenesis associated with smoking. We now report that micromolar concentrations of iron or free fatty acid are not toxic to cultured human lung fibroblasts. However, when combined, the same low concentrations of iron and free fatty acid exert synergistic toxicity. Furthermore, the combination of free fatty acid and iron is highly mutagenic, inducing almost as many selectable mutations in the gene for hypoxanthine/guanine phosphoribosyl transferase as does benzo[a]pyrenediolepoxide, a class I carcinogen generated from benzo[a]pyrene present in cigarette smoke. The combination of free fatty acid and iron also promotes transformation of NIH 3T3 cells into an anchorage-independent phenotype. We conclude that free fatty acids in tobacco smoke may be important contributors to both the pulmonary damage and the carcinogenesis associated with smoking.     

The genotoxicity of alpha particles in human embryonic skin fibroblasts

Cell inactivation and induced mutation frequencies at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus have been measured in cultured human fibroblasts (GM10) exposed to alpha particles from 238Pu (LET at the cell surface was 100 keV/microns) and 250 kVp X rays. The survival curves resulting from exposure to alpha particles are exponential. The mean lethal dose, D0, is approximately 1.3 Gy for X rays and 0.25 Gy for alpha particles. As a function of radiation dose, mutation induction at the HGPRT locus was linear for alpha particles whereas the X-ray-induced mutation data were better fitted by a quadratic function. When mutation frequencies were plotted against the log of survival, mutation frequency at a given survival level was greater in cells exposed to alpha particles than to X rays.     

Cytotoxic effects of colicins E1–E5 and K on hamster fibroblasts

As a further model of mammalian tissue cells, Chinese hamster fibroblasts of the stable line V79 were used to cheek the cytotoxic effects of colicins. The efficiency of plating of cells treated with colicins E1–E5 and with colicin K was followed. The V79 cells were, in general, poorly sensitive to colicins; only colicins E1, E3 and E5. lowered the number of colony-forming cells to some degree. Again, a different action of colicins E1 and K (which is the same in bacteria) was found in eukaryotic cells.     

Total-body irradiation with high-LET particles: acute and chronic effects on the immune system

Although the immune system is highly susceptible to radiation-induced damage, consequences of high linear energy transfer (LET) radiation remain unclear. This study evaluated the effects of 0.1 gray (Gy), 0.5 Gy, and 2.0 Gy iron ion (56Fe(26)) radiation on lymphoid cells and organs of C57BL/6 mice on days 4 and 113 after whole body exposure; a group irradiated with 2.0 Gy silicon ions (28Si) was euthanized on day 113. On day 4 after 56Fe irradiation, dose-dependent decreases were noted in spleen and thymus masses and all major leukocyte populations in blood and spleen. The CD19(+) B lymphocytes were most radiosensitive and NK1.1(+) natural killer (NK) cells were most resistant. CD3(+) T cells were moderately radiosensitive and a greater loss of CD3(+)/CD8(+) T(C) cells than CD3(+)/CD4(+) T(H) cells was noted. Basal DNA synthesis was elevated on day 4, but response to mitogens and secretion of interleukin-2 and tumor necrosis factor-alpha were unaffected. Signs of anemia were noted. By day 113, high B cell numbers and low T(C) cell and monocyte percents were found in the 2.0 Gy 56Fe group; the 2.0 Gy 2)Si mice had low NK cells, decreased basal DNA synthesis, and a somewhat increased response to two mitogens. Collectively, the data show that lymphoid cells and tissues are markedly affected by high linear energy transfer (LET) radiation at relatively low doses, that some aberrations persist long after exposure, and that different consequences may be induced by various densely ionizing particles. Thus simultaneous exposure to multiple radiation sources could lead to a broader spectrum of immune dysfunction than currently anticipated.     

Fibrin clot retraction by human skin fibroblasts: effects of ADP and thrombin.

Thrombin stimulated human skin fibroblasts to retract fibrin clots. When Bothrops marajoensis thrombinlike enzyme was substituted for thrombin, no retraction occurred. Fibroblasts were found to contain 12 nmole of ATP and 3.6 nmole of ADP/mg of protein, a value closely resembling that of nonmetabolic adenine nucleotides in platelets. Thrombin caused neither release of adenine nucleotides from the suspension of fibroblasts harvested enzymatically nor did addition of ADP stimulate fibroblasts to retract fibrin clots.     

The effects of monensin on membrane lipids of cultured human skin fibroblasts

We have investigated the effects of monensin, a monovalent cationophore, on the metabolism of neutral lipids, fatty acids, ceramide and phospholipids in cultured human skin fibroblasts. Treatment with 1 microM monensin for 18 h reduced the cellular cholesterol ester content to less than one-third of untreated cells, and incorporation of [3H]acetate into cholesterol ester was also reduced, to less than one-fifth. Concomitantly, a greater conversion of [3H]acetate into free cholesterol occurred. There was a moderate increase in free fatty acids, but no change in triacylglycerol content, although the content of the latter appeared to increase in the presence of fetal calf serum in the culture medium. Phosphatidylcholine decreased in content and phosphatidylserine increased among the phosphatides, but ceramide remained unchanged after monensin treatment. These findings suggest that monensin influences the metabolic interrelationships of structural lipids in fibroblasts.     

The stimulatory effects of alpha1-adrenergic receptors on TGF-beta1, IGF-1 and hyaluronan production in human skin fibroblasts

Skin fibroblasts modulate tissue repair, wound healing and immunological responses. Adrenergic receptors (ARs) mediate important physiological functions, such as endocrine, metabolic and neuronal activity. In this study, the expression α1A-ARs in human skin fibroblasts is examined and verified. Regulatory effects of α1-agonist cirazoline on cell migration and the production of transforming growth factor β1 (TGF-β1), insulin-like growth factor 1 (IGF-1), hyaluronan (HA), fibronectin and procollagen type I carboxy-terminal peptide (PIP) by human skin fibroblasts are assessed and validated. α1A-AR mRNA and protein were found in human skin fibroblasts WS1. Exposure of cirazoline doubled skin fibroblast migration and the increase in cell migration was attenuated by α1-antagonist prazosin. TGF-β1 mRNA and production were enhanced after exposure to cirazoline and IGF-1 production was also increased after treatment with cirazoline. Exposure to cirazoline also enhanced HA and PIP production. The increases in TGF-β1, IGF-1, HA and PIP production were partially abolished in fibroblasts transfected with α1A-AR short interfering RNAs, indicating that α1A-ARs are involved in the cirazoline-induced increases in TGF-β1, IGF-1, HA and PIP production. Thus, α1A-ARs are stably expressed and stimulate cell migration and TGF-β1, IGF-1, HA and PIP production in human skin fibroblasts. Moreover, TGF-β1, IGF-1, HA and PIP production and the cell migration of human skin fibroblasts are possibly modulated by natural catecholamines produced by the endocrine system or sympathetic innervation, which could directly or indirectly participate in cytokine secretion, fibroblast migration and matrix production of wound healing in the skin.     

Cytotoxic and mutagenic effects of emodin on cultured mouse carcinoma FM3A cells

Employing a suspension culture of a mouse mammary carcinoma cell line, FM3A cells, the cytotoxicity and induced mutagenicity of emodin (EM) were examined and compared with those of 2-hydroxy-emodin (2-OH-EM), which was identified as an active form of EM in the Ames/microsomes assay. EM was cytotoxic to FM3A cells in concentrations of 1-10 micrograms/ml, and induced 6-thioguanine-resistant (6TGr) mutation. 2-OH-EM was a little more toxic than EM, but induced little mutation.     

Synthesis and regulation of C1 inhibitor in human skin fibroblasts

Proteins of the C1 complex, C1q, C1r, and C1s, of the classical pathway of complement activation are known to be synthesized in human skin fibroblasts. Using metabolic labeling with [35S]methionine, immunoprecipitation, and SDS-PAGE, we demonstrate that human skin fibroblasts synthesize and secrete C1 inhibitor with an apparent molecular mass of 78 kDa in the cell lysate and 102 kDa in the extracellular medium. This C1 inhibitor had the capacity to bind activated C1s. Fibroblasts synthesized 30- to 50-fold more C1 inhibitor than was synthesized in monocytes. As previously reported, fibroblasts also synthesized C1r and C1s. IFN-gamma, IFN-beta 1, and TNF had significant, but distinct, effects on synthesis of C1 inhibitor, C1r, and C1s. Incubation of the cells with IFN-gamma, 1000 U/ml, for 24 h induced increases in the synthesis of C1 inhibitor, C1r, and C1s by 4.2-, 1.9- and 1.6-fold, respectively. IFN-beta 1 had effects similar to IFN-gamma, although smaller in magnitude. TNF, 12.5 ng/ml, induced increases in the synthesis of C1 inhibitor, C1r, and C1s by 1.5-, 1.4- and 2.6-fold. IL-1, IFN-beta 2 (IL-6), and LPS did not affect synthesis of C1 inhibitor, C1r, or C1s. Fibroblasts are present in large amounts in most tissues. Synthesis of C1 inhibitor, C1r, and C1s by these cells could provide a source of these important proteins in body tissues. In addition, fibroblasts should be a good model for the in vitro study of genetic diseases involving the synthesis of these proteins.     

Genotoxic effects of high-energy iron particles in human lymphoblasts differing in radiation sensitivity.

The effects of (56)Fe particles and (137)Cs gamma radiation were compared in TK6 and WTK1 human lymphoblasts, two related cell lines which differ in TP53 status and in the ability to rejoin DNA double-strand breaks. Both cell lines were more sensitive to the cytotoxic and clastogenic effects of (56)Fe particles than to those of gamma rays. However, the mutagenicity of (56)Fe particles and gamma rays at the TK locus was the same per unit dose and was higher for gamma rays than for (56)Fe particles at isotoxic doses. The respective RBEs for TK6 and WTK1 cells were 1.5 and 1.9 for cytotoxicity and 2.5 and 1.9 for clastogenicity, but only 1 for mutagenicity. The results indicate that complex lesions induced by (56)Fe particles are repaired less efficiently than gamma-ray-induced lesions, leading to fewer colony-forming cells, a slightly higher proportion of aberrant cells at the first division, and a lower frequency of viable mutants at isotoxic doses. WTK1 cells (mutant TP53) were more resistant to the cytotoxic effects of both gamma rays and (56)Fe particles, but showed greater cytogenetic and mutagenic damage than TK6 cells (TP53(+)). A deficiency in the number of damaged TK6 cells (a) reaching the first mitosis after exposure and (b) forming viable mutants can explain these results.     

The effects of amphotericin B on lipid metabolism in cultured human skin fibroblasts

Summary Amphotericin B inhibits hydroxymethylglutarylcoenzyme A (HMGCoA) reductase activity and incorporation of [³H]acetate into sterols and fatty acids of human skin fibroblasts. Delivery of cholesterol to cells via the low density lipoprotein receptor pathway is not altered. The effects of amphotericin B on cell lipid metabolism are partially reversed by both potassium and insulin. Portions of this work were presented at the Southern Regional Meeting of the American Federation for Clinical Research, January 1983.     

Yield of single- and double-strand breaks and nucleobase lesions in fully hydrated plasmid DNA films irradiated with high-LET charged particles

We measured the yield and spectrum of strand breaks and nucleobase lesions produced in fully hydrated plasmid DNA films to determine the linear energy transfer (LET) dependence of DNA damage induced by ion-beam irradiation in relation to the change in the atomic number of ions. The yield of isolated damage was revealed as a decrease in prompt SSBs with increasing LET of He(2+), C(5+,6+) and Ne(8+,10+) ions. On the other hand, the yields of prompt DSBs increased with increasing ion LET. SSBs were additionally induced in ion-irradiated DNA film by treatment with two kinds of base excision repair proteins (glycosylases), Nth and Fpg, indicating that base lesions are produced in the hydrated DNA film. This result shows that nucleobase lesions are produced via both chemical reactions with diffusible water radicals, such as OH radicals, and direct energy deposition onto DNA and the hydrated water layer. Nth-sensitive sites deduced to be pyrimidine lesions, such as 5,6-dihydrothymine (DHT), showed a relatively larger yield than Fpg-sensitive sites deduced to be purine lesions, such as 7,8-dihydro-8-oxo-2'deoxyguanine (8-oxoGua), for all ion exposures tested. The yield of SSBs or DSBs observed by enzyme treatment decreased noticeably with increasing LET for all tested ions. These results indicated that higher-LET ions preferentially produce a complex type of damage that might compromise the activities of the glycosylases used in this study. These findings are biologically important since, under cell mimicking conditions, persistent DNA damage occurs in part due to direct energy deposition on the DNA or hydrated water shell that is specifically induced by dense ionization in the track.     

Absence of hyaluronidase in cultured human skin fibroblasts.

The in vitro degradation of [³⁵S]chondroitin sulfate was investigated in human fibroblasts and rat liver. In rat liver, preparations of chondroitin sulfate were shown to be degraded by the concerted action of endoglycosidase and exoglycosidases. However, with human skin fibroblast preparations, hyaluronidase activity was not detected and chondroitin sulfate was degraded by exoglycosidase action.     

Respective effects of glucose and glutamine on the glutamine synthetase activity of human skin fibroblasts

Human A431 carcinoma cell line is known to have 30 fold amplified epidermal growth factor receptor (EGF-R) gene. We have studied the effect of steroid hormone dexamethasone (DEX) and protein synthesis inhibitor cycloheximide (CHX) on the expression of EGF-R gene in this cell line. DEX treatment and protein synthesis inhibition by CHX treatment cause a rapid 3 to 4 fold increase in the level of EGF-R mRNA and combined treatment of the above two agents have less than additive effect. It appears that mRNA for EGF-R accumulate within the cell during protein synthesis inhibition and upon removal of CHX, gets translated into EGF-R specific protein as judged by immuno-dot assay. We did not observe the phenomenon of super induction nor much of an additive effect under condition of combined DEX and CHX treatment.Abbreviations EGF-R Epidermal Growth Factor Receptor - DEX Dexamethasone - CHX Cycloheximide