Potentiating effects of organomercuries on clastogen-induced chromosome aberrations in cultured Chinese hamster cells

Mercury compounds are among the most serious environmental pollutants. In this communication, the potentiating effects of organic and inorganic mercuries on clastogen-induced chromosome aberrations were studied in Chinese hamster CHO K1 cells. Post-treatment with monoalkylated mercuries — methyl mercuric chloride (MeHgCl) and ethyl mercuric chloride (EtHgCl) - increased the number of breakage-and exchange-type aberrations induced by 4-nitroquinoline 1-oxide (4NQO) and methyl methanesulfonate. With the DNA crosslinking agents mitomycin C (MMC) and cisplatin, MeHgCl enhanced both types of aberrations while EtHgCl enhanced breakage-type aberrations only. Since these monoalkylated mercuries did not show clastogenic effects by themselves under the present experimental conditions, the increases in chromosome aberrations were not additive. Dialkylated mercuries (dimethyl mercury and diethyl mercury) and inorganic mercuries (HgCl and HgCl₂) did not show any potentiating effects.

When MMC- or 4NQO-treated cells were post-treated with MeHgCl during the G1 phase, both breakage- and exchange-type aberrations were enhanced. Treatment with EtHgCl during the G1 phase also enhanced both types of aberrations induced by 4NQO. With MMC, however, G1 treatment with EtHgCl did not show any potentiating effect. MeHgCl and EtHgCl treatments during the G2 phase enhanced breakage-type aberrations only.

Based on these results, the following possible mechanisms for potentiation of clastogenicity by monoalkylated mercuries were suggested; (1) they interfere with repair of base lesions induced by 4NQO and MMS during the pre-replicational stage, thereby increasing unrepaired DNA lesions which convert into DNA double-strand breaks in S phase, (2) MeHgCl (but not EtHgCl) also inhibits repair of crosslinking lesions during the pre-replicational stage, and (3) their G2 effects enhance breakage-type aberrations only.     

A comparative study of the toxicity of mercury dichloride and methylmercury, assayed by the Frog Embryo Teratogenesis Assay--Xenopus (FETAX)

The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a powerful and flexible bioassay that makes use of the embryos of the anuran amphibian Xenopus laevis. The FETAX can detect xenobiotics that affect embryonic development, when mortality, teratogenicity and growth inhibition are used as endpoints. The FETAX was used to compare the embryotoxic and teratogenic potentials of two chemical species of mercury, inorganic mercury(II) chloride (HgCl2) and organic methylmercury chloride (MeHgCl). A higher toxicity of MeHgCl (the estimated median lethal concentration [LC50] and median teratogenic concentration [TC50] were 0.313microM and 0.236microM, respectively) over HgCl2, with estimated LC50 and TC50 values of 0.601microM and 0.513microM, respectively). On the basis of these results, HgCl2 and MeHgCl can be classified as "slightly teratogenic compounds", as the ratio of LC50/TC50 is less than 1.5. There was a significant deviation from the commonly described monotonic behaviour of the concentration-response curves, suggesting a hormetic effect of both species of mercury. Uptake experiments, followed by neutron activation analysis, showed a higher incorporation of mercury in embryos exposed to MeHgCl compared with those exposed to HgCl2. Interestingly, Hg- exposed embryos showed a higher content of selenium and zinc than did control embryos.     

Mercury chloride increases hepatic alanine aminotransferase and glucose 6-phosphatase activities in newborn rats in vivo

This work investigated the in vivo and in vitro effects of HgCl2 and ZnCl2 on metabolic enzymes from tissues of young rats to verify whether the physiological and biochemical alterations induced by mercury and prevented by zinc are related to hepatic and renal glucose metabolism. Wistar rats received (subcutaneous) saline or ZnCl2 (27 mg/kg/day) from 3 to 7 days old and saline or HgCl2 (5.0 mg/kg/day) from 8 to 12 days old. Mercury exposure increased the hepatic alanine aminotransferase (～6-fold) and glucose 6-phosphatase (75%) activity; zinc pre-exposure prevented totally and partially these mercury alterations respectively. In vitro, HgCl2 inhibited the serum (22%, 10 μM) and liver (54%, 100 μM) alanine aminotransferase, serum (53%) and liver (64%) lactate dehydrogenase (10 μM), and liver (53%) and kidney (41%) glucose 6-phosphatase (100 μM) from 10- to 13-day-old rats. The results show that mercury induces distinct alterations in these enzymes when tested in vivo or in vitro as well as when different sources were used. The increase of both hepatic alanine aminotransferase and glucose 6-phosphatase activity suggests that the mercury-exposed rats have increased gluconeogenic activity in the liver. Zinc prevents the in vivo effects on metabolic changes induced by mercury.     

Inhibition of renal Na(+)-Pi cotransporter by mercuric chloride: role of sulfhydryl groups.

We studied the role of sulfhydryl groups in Na(+)-Pi cotransport across the renal brush border membrane (BBM), using HgCl2, an agent which penetrates membranes freely. HgCl2 inhibited the initial Na(+)-dependent 32Pi transport in a dose-dependent manner (IC50 = 54 microM). Na(+)-independent transport was not affected. The inhibitory effect persisted under Na+ equilibrium-exchange conditions. Additionally, HgCl2 had no effect on the diffusional uptake of 22Na up to 1 min incubation. Exposure to HgCl2 had no effect on vesicle integrity as determined by osmotic shrinking experiments. BBM vesicle (BBMV) volume, determined by D-glucose equilibrium uptake, was not affected at low HgCl2 concentrations, but decreased at higher concentrations (greater than 100 microM). Vesicle volumes, determined by flow cytometry, were not changed after exposure to HgCl2. Kinetic studies showed a reduction in the apparent Vmax for Pi transport from 1.40 +/- 0.13 to 0.75 +/- 0.19 nmoles/mg protein/5 sec, without a significant change in the apparent Km. In protection studies, dithiothreitol (DTT) completely protected against inhibition, but Pi, phosphonoformic acid (PFA), and Na+ gave no protection. The data suggest that sulfhydryl groups are essential for the function of Na(+)-Pi cotransporter of renal BBM.     

Effects of Different Calcium, Magnesium, and Zinc Concentrations Supplemented on Hepatopancreatic Cell Proliferation of Kuruma Prawn, Penaeus japonicus

The effects of different calcium (Ca(2+)), magnesium (Mg(2+)), and zinc (Zn(2+)) concentrations supplemented on hepatopancreatic cell proliferation of kuruma prawn, Penaeus japonicus was studied. The culture system consists of medium 199 (M 199) supplemented with 0.060 mol/L NaCl, 1.011 g/L glucose, 1,000 UI/ml penicillin, 1,000 μg/ml treptomycin, heat inactivated fetal calf serum (FCS) 20% for primary cell culture and 10% for subculture. The RNA/DNA ratio of the cell cultures was measured. The results show that the cell division of hepatopancreatic cells of P. japonicus was enhanced by the optimal concentration of inorganic salt (Ca(2+), 1.0 g/L; Mg(2+), 5.0 g/L; Zn(2+), 80 μg/L). The hepatopancreatic cell culture system and improved culture conditions described here will be very useful for in vitro experiments to study viruses responsible for infections in shrimp leading to tremendous economic losses.     

Genotoxicity of cadmium chloride in human lymphocytes evaluated by the comet assay and cytogenetic tests

Peripheral blood lymphocytes were tested in vitro for genotoxic effects of cadmium chloride. Whole blood samples of four healthy, non-smoking subjects were preincubated with CdCl2 in concentrations of 10(-4), 10(-3), and 5 . 10(-3) mol/L for three hours before the cells were assessed for DNA-damage using the single cell alkaline gel electrophoresis assay (comet assay) or cultivated for chromosomal aberrations (CA), sister chromatid exchanges (SCE), and the micronucleus (MN) test. The comet assay showed notable interindividual differences. The results of the cytogenetic tests showed an increase in the frequency of CA, MN, and SCE with CdCl2 in the treated cultures, yet none was able to show a correlation between concentrations of cadmium chloride and the frequency of damages. The MN slides were stained with Giemsa and with DNA fluorochrome 4', 6'-diamidino-2-phenylindole (DAPI). The frequency of MN in slides stained with DAPI was significantly higher than in those stained with Giemsa, which might be due to an underestimation of small micronuclei in Giemsa-stained slides.     

Lipid peroxidation in liver of rats administrated with methyl mercuric chloride

Parenteral administration of methyl mercuric chloride (MMC, CH₃HgCl) to rats enhanced lipid peroxidation in liver of rats, as measured by the thiobarbituric acid reaction for malondialdehyde (MDA) in fresh tissue homogenates. After sc injection of CH₃HgCl (5 mg/kg body wt), MDA concentration in liver became significantly increased at 24 h and further increased at 48 h. Dose-response studies were carried out with male albino rats of the Fisher-344 strain (body wt 170–280 g) injected with 3 or 5 mg Hg/kg as CH₃HgCl and sacrificed after 24 h. In time-response studies, animals were administered 5 mg Hg/kg as CH₃HgCl and sacrificed after 24 and 48 h. Studies in the authors’ laboratory have shown that (1) mercury is accumulated in liver; (2) concentration of MDA is increased in liver of CH₃HgCl-treated rats; (3) severity of hepatotoxicity is generally proportional to the elevation of MDA concentration, based upon the dose-effect relationships observed after administration of CH₃HgCl to rats. The results of this study implicate that the lipid peroxidation is one of the molecular mechanisms for cell injury in acute CH₃HgCl poisoning.     

Macrophages and methylthio groups in lymphocyte proliferation

Macrophages are shown to replace methylthio disulfides in supporting in vitro proliferation of three cell lines previously characterized as methylthio-dependent. Macrophages have the capacity to generate methylthio groups from methylthioadenosine. It is hypothesized that macrophages stimulate cell proliferation both in normal immune systems and in certain cancers by providing an abundance of methylthio groups. Fetal calf serum is shown to contain methylthio groups. It appears that, in cell cultures containing fetal calf serum, sulfhydryl compounds stimulate cell proliferation by making the methylthio groups in the serum available to the cells.     

HgCl2对泥鳅精子运动的影响

为了检测不同浓度的HgCl2对泥鳅Misgurnus anguillicaudatus精子运动的影响,以泥鳅精子为实验材料,用含终浓度分别为0(对照)、1、5、10、15和20 μmoL/L的HgCl2待测液分别孵育0、2、4和6 h后激活,激活后立即在显微镜(Olympus IX81)下观察精子运动参数.为进一步探讨HgCl2对泥鳅精子运动影响的机制,用终浓度为20μmol/L的HgCl2保存液孵育泥鳅精子10 min,以含终浓度分别为0、0.1、1、10 mmol/L的2-巯基乙醇和20μmol/LHgCl2混合液为激活液激活.激活后立即在显微镜下观察精子运动,发现2-巯基乙醇町逆转HgCl2对泥鳅精子的抑制作用,为探讨HgCl2对泥鳅精子运动影响的可能机制提供参考.     

Induction of umbelliferone in sweet potato cell suspension culture using mercuric chloride

HgCl₂ was used at up to 10 mg l^–1 as an elicitor of phytoalexins in sweet potato (Ipomoea batatas (L.) Lam. cv Centennial) cell suspension cultures. Maximum stimulation of a coumarin compound was after one day of exposure using 1 mg HgCl₂ l^–1. The compound was identified by HPLC and GC-MS analyses as 7-hydroxycoumarin (umbelliferone).     

Epithelial cell differentiation in organotypic cultures of fetal rat lung

The purpose of this investigation was to examine the suitability of an organotypic lung-cell culture model for the study of factors influencing fetal lung-cell differentiation. It has been reported that the use of carbon-stripped (hormone-depleted) bovine fetal calf serum in monolayer cell cultures of fetal rat lung prevents continued epithelial cell differentiation in vitro. In this study, organotypic cultures of fetal rat lung cells taken at day 20 of gestation (late canalicular stage) were prepared with a carbon-stripped medium. These organotypic cultures were examined by light, scanning, and transmission electron microscopy for comparison with controls prepared with unstripped bovine fetal calf serum. Highly organized three-dimensional tubular epithelial structures resembling saccules of immature lung were observed within the gelatin sponge matrix. Morphometric analysis of day 20 carbon-stripped samples revealed that 74.6% of the epithelial cells in the tubular structures contained osmiophilic lamellar bodies characteristic of type II pneumonocytes. Control specimens had 71.2% cells with lamellar bodies and did not differ significantly from the experimental group. These data are similar to those obtained with organ cultures of fetal rat lung but are in contrast to findings with monolayer culture systems. The observations of this study suggest that 1) the hormones extracted from bovine fetal calf serum by carbon-stripping are not solely responsible for the continued fetal lung cell differentiation observed in vitro, and 2) that spatial relationships between lung cells in vitro may be a significant factor in the control of differentiation.     

Utilization of whey fractions as a substitute for fetal calf serum in culture media

Summary A dairy by-product was used in cell line cultures as a replacement for fetal calf serum (FCS). Various fractions were prepared from whey. All fractions have the ability to stimulate DNA synthesis in human and mouse 3T3 fibroblast and hybridoma cultures. They also allow hybridoma proliferation in the long term using only 0.5% FCS. In these latter experiments, heated and ultrafiltered fractions were more effective than 10% fetal calf serum.     

Effects of serum and glucose on the proliferation of different types of cells of the human embryonic pancreas in primary culture

We have utilized primary cultures of free-floating clusters obtained from pancreata of human cadaveric fetuses. Clusters have been incubated for 10 days in RPMI-1640 medium with 3H-thymidine and various concentrations of fetal calf serum and glucose. Viability of clusters was assessed by insulin radioimmunoassay. Cell reproduction was investigated by radioautography. We have revealed 2 populations of proliferating epithelial cells in clusters: the minor population of cells of duct-like structures and the major population of other epithelial cells. Rate of accumulation of labelled cells in both populations in clusters of 17-18-week-old fetuses was significantly higher than in clusters of 23-24-week-old fetuses. Proliferation of cells of duct-like structures was regulated by serum but not by glucose, whereas proliferation of other epithelial cells depended on both serum and glucose concentrations.     

Identification of media supplements that improve the viability of primarily cell cultures ofCrassostrea gigas oysters

Media supplements have been investigated for their influence on the viability of primary cell cultures from the heart ofCrassostrea gigas oysters. Soluble factors of vertebrate origin were tested, belonging to five families of supplements that had proven to increase the viability of insect and mammal cell cultures. Using two-level complete factorial assays, factors and mutual interactions were screened within each family with a MTT reduction assay. Results pointed out the positive influence of hormones, growth factor, antioxidants and lipids on the mitochondrial metabolism of oyter's heart cells. Consequently, a new concentrated complex supplement was developed. At 10% (v/v) final concentration in modified Leibovitz L-15 medium, it increases by 30% the cellular viability of one-week old cultures as compared with non-supplemented medium, a similar improvement as the one obtained with 10% (v/v) fetal calf serum. Combined with fetal calf serum, this new supplement doubles the cellular viability of one-week old cultures and allows networks of cardiomuscular cells to be maintained functional over three monthsin vitro.Abbreviations MTT-3 (4,5-dimethylthiazol;-2-yl)2,5-diphenyl-tetrazolium bromide - FCS fetal calf serum     

Thermolabile inhibiting proliferation of connective tissue cell activity from platelets

The action of a platelet extract (PE) and a heat-inactivated PE on 3T3 cells proliferation has been investigated. The heat-inactivated PE and platelet poor plasma (PPP) together can promote cell proliferation like fetal calf serum. At the same time PE inhibits proliferation of the cell culture stimulated by 5% fetal calf serum, whereas the heat-inactivated PE does not. Although the actions of 2% PE and 2% heat-inactivated PE on the cells incubated with PPP are equal, the stimulating effect of a 10% heat-inactivated PE is higher than that of a 10% PE. Thus, the inhibitor acts only at high concentrations. The role of the platelet-derived inhibitor in the limiting of extensive cell proliferation during vessel wall injury repair is discussed.     

Inhibition of cell proliferation and protease activity by cartilage factors and heparin

Proliferating rat smooth muscle cells and fibroblasts have membrane-associated protease activity. High concentrations of heparin inhibited membrane-associated protease activity and cell proliferation, while low concentration of heparin promoted smooth muscle cell proliferation. The inhibition of protease activity and proliferation was abolished when heparin was treated with protamine sulfate or when acid treated fetal calf serum was used. Heparin required the presence of an acid labile factor(s) in serum for the inhibition of protease activity and proliferation. Heparin and antithrombin III in the presence of acid-treated fetal calf serum did not inhibit cell proliferation or protease activity. Cartilage factors isolated from bovine nasal cartilage containing trypsin inhibitory activity, but not papain inhibitory activity, inhibited rat smooth muscle and fibroblast proliferation and surface associated protease activity. The cartilage factors did not require acid-labile components in the fetal calf serum for the inhibitory activity. The inhibitory activity due to heparin and cartilage factors was not permanent under our experimental condition. Protein synthesis was not inhibited by heparin or the cartilage factors. In rat smooth muscle cells and fibroblasts, the expression of surface-associated protease activity was related to the proliferative state of the cells. Surface protease activity was only present on proliferating cells. When surface protease activity was inhibited by high concentrations of heparin in the presence of an acid-labile serum component(s) or cartilage factors, cell proliferation was also inhibited.     

Enhancement of osteoblast proliferative capacity by growth factor-like molecules in bear serum

The use of animal serum in cell culture is vital for providing the nutrient factors required to promote proliferation and function. Fetal calf serum has become the preferred choice because of its abundance, reasonable cost, and ability to sustain human cells in vitro. Although a wide variety of serum sources have been tested and used, little is known about the ability of serum obtained from the American black bear (Ursus americanus) to support human cell growth in culture. The American black bear, an animal comparable in size to humans, is unique in that it hibernates for mo at a time but does not experience extensive bone loss normally associated with extended immobility. The aim of this study was to analyze the effect of bear serum on human osteoblast cultures. We discovered that three of the eight bear serum samples induced significantly higher proliferation rates in osteoblasts than did fetal calf serum over a 24-h period. Osteoblasts incubated in bear serum displayed higher messenger ribonucleic acid levels for phenotype markers osteocalcin and type I collagen than did those incubated in fetal calf serum. The mitogenic activity of the bear serum was reduced when heated at 56 degrees C for 30 min before use in culture. The molecular weight of the mitogenic factors was found to be primarily greater than 50 kDa. The present work demonstrates the capability of serum from American black bears to support human osteoblast proliferation in vitro.     

Single-strand breaks, cell cycle arrest and apoptosis in HL-60 and LLCPK1 cells exposed to 1,2-dibromo-3-chloropropane

We investigated 1,2-dibromo-3-chloropropane (DBCP)-induced DNA damage, cell cycle alterations and cell death in two cell lines, the human leukemia HL-60 and the pig kidney LLCPK₁, both of which are derived from potential target sites for DBCP-induced toxicity. DBCP (30–300 µmol/L) caused a concentration-dependent increase in the levels of DNA single-strand breaks in both cell lines as well as in cultured human renal proximal tubular cells. After extended DBCP exposure in LLCPK₁ cells (100 µmol/L, 30 h), the level of DNA breaks returned almost to control values. Incubation for 48 h showed a clear reduction of growth with DBCP concentrations as low as 10 µmol/L. Flow cytometric analysis showed that DBCP (1–10 µmol/L) exposure for 24 h caused an accumulation of LLCPK₁ cells in the G₂/M-phase. In HL-60 cells the accumulation in G₂/M-phase was less marked, and at higher concentrations the cells accumulated in S-phase. Flow cytometric studies of HL-60 and LLCPK₁ cells exposed to 100–500 µmol/L DBCP showed increased number of apoptotic cells/bodies with a lower DNA content than that of the G₁ cells. Microscopic studies revealed that there were increased numbers of cells with nuclear condensation and fragmentation, indicating that apoptosis was the dominant mode of death in these cell lines, following exposure to DBCP. The characteristic ladder pattern of apoptotic cells was observed when DNA from DBCP-treated HL-60 cells and LLCPK₁ cells was electrophoresed in agarose. The finding that DBCP can cause an accumulation of cells in G₂/M-phase and induce apoptosis in vitro may be of importance for the development of DBCP-induced toxicity in vivo.     

Endogenous fragment of hemoglobin, neokyotorphin, as cell growth factor

It is shown that neokyotorphin (the -globin fragment 137–141) stimulates proliferation of normal cells (murine embryonic fibroblasts, red bone marrow and spleen cells) and tumor cells (murine melanoma and transformed fibroblasts L929) in the absence or in the presence of fetal bovine serum. In contrast to serum deprivation conditions, the ability to potentiate L929 cell growth in the presence of fetal serum is strongly cell density dependent. The peptide also enhances the viability of L929 cells, murine embryonic fibroblasts and of the primary cultures of murine red bone marrow cells and splenocytes under serum-deprivation conditions for at least 72 h. The results of flow cytometry analysis suggest that the effect of neokyotorphin on survival of L929 cells in serum-free culture medium is due to maintenance of cell proliferation in the absence of growth factors. Along with cell cycle progression the peptide induces reversible reduction of L929 cell size.     

Stimulation by glucocorticoids of myoblast growth at low cell densities

Proliferation of rat myoblasts was stimulated by addition of 10(-7) M dexamethasone to cells plated at 25 cells/cm(2). In the presence of fetal calf serum and chick embryo extract, physiological levels of glucocorticoids caused a 3- to 7-fold increase in colony formation. In mass cultures, proliferation of myoblasts was also stimulated by dexamethasone, but fibroblast growth was inhibited. These opposite effects of glucocorticoids on myoblast and fibroblast growth suggest their use in cloning muscle cells from mixed cell populations.