Induction of unscheduled DNA synthesis and micronucleus formation in Syrian hamster embryo fibroblasts treated with cysteine S-conjugates of chlorinated hydrocarbons

S-(chloroethyl)-cysteine (CEC) and S-(1,2-dichlorovinyl)cysteine (DCVO) have been proposed as intermediates in the metabolic transformation of the carcinogens 1,2-dichloroethane and 1,1,2-trichloroethylene. We have tested the ability of CEC and DCVC to induce DNA repair and genotoxic effects at the chromosomal level by comparative assessment of unscheduled DNA synthesis induction and micronucleus formation in Syrian hamster embryo fibroblasts. CEC induced a potent and dose-dependent response in both assays, whereas DCVC treatment resulted in a comparatively weak induction of DNA repair and failed to raise micronucleus formation above control rates. Inhibition of cysteine conjugate \gB-lyase diminished the effect of DCVC, but had no influence on the genotoxicity of CEC either in the unscheduled DNA synthesis or micronucleus assay.Abbreviations AOAA aminooxyacetic acid - CEC S-(chloroethyl)-cysteine; \gB-lyase, cysteine conjugate -lyase - DCE 1,2-dichloroethane - DCVC S(1,2-dichlorovinyl)-cysteine - GSH glutathione - HU hydroxyurea - IBR IBR-modified Dulbecco's Eagle's reinforced medium - MN2 micronuclei/2,000 cells - 4-NQO 4-nitroquinoline-1-oxide - SHE Syrian hamster embryo fibroblasts; ³H-Thd, ³H-thymidine - TCE 1,1,2-trichloroethylene - UDS unscheduled DNA synthesis     

Transforming activities of sodium fluoride in cultured Syrian hamster embryo and BALB/3T3 cells

The transforming activity of sodium fluoride was studied in the SHE and the BALBl3T3 cell culture systems. Initiating and promoting activities were then investigated by means of the orthogonal methodology. Sodium fluoride was found to induce morphological transformation of SHE cells seeded on a feeder layer of X-irradiated cells at high concentrations (75–125 g/ ml). When the cells were seeded in the absence of a feeder-layer, the transformation frequencies increased in a dose-dependent manner with the concentrations of sodium fluoride ranging from 0 to the highly toxic concentration of 200 g/ml. In the BALBl3T3 cell system, sodium fluoride was negative in the standard Kakunaga procedure, while through the experiment designed by table L₈ (2⁷) of the orthogonal method, an initiating-like effect and a weak promoting activity were detected within the concentrations ranging from a 25 g/ ml to a 50 g/ ml concentration which is highly toxic for BALBl3T3 cells. From these results, it is suggested that, besides a genetic mode of action, sodium fluoride could possibly act through a non-genotoxic mechanism.Abbreviations CE cloning efficiency - NaF sodium fluoride - SHE Syrian hamster embryo - TF transformation frequency     

Characterization of an unscheduled DNA synthesis assay with Syrian hamster embryo cells

The Syrian hamster embryo cell transformation assay is widely used for studies of carcinogenesis. The characterization of an unscheduled DNA synthesis (UDS) assay for these cells is reported. Benzo[a]pyrene, aflatoxin B1 and UV light induced UDS in the cells in a dose-dependent manner without exogenous metabolic activity. Nitrosopiperidine induced UDS as well as gene mutations and cell transformation only in the presence of an exogenous metabolic activation system. The utility of this UDS assay with these cells is discussed.     

Induction by modified purines (2-aminopurine and 6-N-hydroxylaminopurine) of chromosome aberrations and aneuploidy in Syrian hamster embryo cells

The modified purines, 2-aminopurine and 6-N-hydroxylaminopurine, are known point mutagens in prokaryotic organisms. 2-Aminopurine is much less potent than 6-N-hydroxylaminopurine in inducing gene mutation in mammalian cells in culture and this corresponds to the relative activity of these two compounds in inducing tumors in rats and neoplastic transformation of Syrian hamster embryo cells in culture. We report here that these modified purines can induce chromosome aberrations, including chromatid gaps, breaks, and exchanges, as well as numerical chromosome changes in Syrian hamster embryo cells. These chromosome mutations occur over the concentration range of chemical needed to induced morphological transformation of the same cells. It is not known how nucleic base analogs induce chromosome mutations; however, this activity must be considered in attempting to understand the mechanism by which these agents induce neoplastic transformation of cells.     

Species specificity affects the choice of S9 preparations for use in the hamster embryo cell transformation system

Summary Before their use as a source of carcinogen-activating enzymes in the hamster embryo cell transformation assay, liver, kidney, lung, and small intestine S9 fractions from Syrian golden hamsters and Sprague-Dawley rats were evaluated for toxicity to hamster embryo target cells. Sprague-Dawley rat liver and kidney S9 were highly toxic to the hamster embryo cells (90 to 100%). When retested at lower concentrations these tissue fractions were still quite toxic (up to 75%). In contrast, hamster liver and kidney S9 were considerably less toxic (14 to 25%). The S9 preparations were also evaluated for their ability to metabolizeN-2-acetylaminofluorene to 2-aminofluorene andN-hydroxy-acetylamino-fluorene, products that transform hamster embryo cells. Large amounts ofN-hydroxy-acetylaminofluorene were formed in the presence of preparations from hamster liver and small intestine, whereas kidney and lung S9 fractions were considerably less active. No detectable levels ofN-hydroxy-acetylaminofluorene were formed after incubation ofN-2-acetylaminofluorene with any of the rat S9 preparations. High levels of deacetylase activity were found in hamster liver and small intestine S9 fractions, at least eightfold higher than those obtained from equivalent rat preparations. Hamster kidney and lung S9 fractions showed low levels of deacetylase activity. There was no detectable activity in equivalent preparations from rats. When tested withN-2-acetylaminofluorene in the hamster embryo cell clonal transformation system, transformed colonies were obtained with hamster liver S9, with and without an external NADPH-generating system. This work was supported by Contract N01-CO-75380 with the National Cancer Institute, NIH, Bethesda, MD 20205.     

Species specificity affects the choice of S9 preparations for use in the hamster embryo cell transformation system

Before their use as a source of carcinogen-activating enzymes in the hamster embryo cell transformation assay, liver, kidney, lung, and small intestine S9 fractions from Syrian golden hamsters and Sprague-Dawley rats were evaluated for toxicity to hamster embryo target cells. Sprague-Dawley rat liver and kidney S9 were highly toxic to the hamster embryo cells (90 to 100%). When retested at lower concentrations these tissue fractions were still quite toxic (up to 75%). In contrast, hamster liver and kidney S9 were considerably less toxic (14 to 25%). The S9 preparations were also evaluated for their ability to metabolize N-2-acetylaminofluorene to 2-aminofluorene and N-hydroxy-acetylaminofluorene, products that transform hamster embryo cells. Large amounts of N-hydroxy-acetylaminofluorene were formed in the presence of preparations from hamster liver and small intestine, whereas kidney and lung S9 fractions were considerably less active. No detectable levels of N-hydroxy-acetylaminofluorene were formed after incubation of N-2-acetylaminofluorene with any of the rat S9 preparations. High levels of deacetylase activity were found in hamster liver and small intestine S9 fractions, at least eightfold higher than those obtained from equivalent rat preparations. Hamster kidney and lung S9 fractions showed low levels of deacetylase activity. There was no detectable activity in equivalent preparations from rats. When tested with N-2-acetylaminofluorene in the hamster embryo cell clonal transformation system, transformed colonies were obtained with hamster liver S9, with and without an external NADPH-generating system.     

Cis-platinum(II) diamine dichloride causes mutation, transformation, and sister-chromatid exchanges in cultured mammalian cells.

The anti-tumor agent cis-platinum(II) diamine dichloride caused dose-dependent toxicity in V79 Chinese hamster cells and in secondary Syrian hamster embryo cells. Chromosome aberrations were induced and positive dose--response relationships were observed for induction of sister-chromatid exchanges and 6-thioguanine-resistant mutations in V79 cells and morphologic transformation of secondary Syrian hamster embryo cells. The findings suggest that this chemical is a potential human carcinogen.     

Photo catalogue for the classification of cell colonies in the Syrian hamster embryo (SHE) cell transformation assay at pH 7.0

This catalogue is a display of Syrian hamster embryo (SHE) cell colony photos representative of the cell transformation assay (CTA) carried out at pH 7.0. It is intended as a visual aid for the identification and the scoring of cell colonies in the conduct of the assay. A proper training from experienced personnel together with the protocol reported in this issue and the present photo catalogue will support method transfer and consistency in the assay results.     

Photo catalogue for the classification of cell colonies in the Syrian hamster embryo (SHE) cell transformation assay at pH 6.7

This catalogue is a display of Syrian hamster embryo (SHE) cell colony photos representative of the cell transformation assay (CTA) carried out at pH 6.7. It is intended as a visual aid for the identification and the scoring of cell colonies in the conduct of the assay. A proper training from experienced personnel together with the protocol reported in this issue and the present photo catalogue will support method transfer and consistency in the assay results.     

A comparative study on neoplastic transformation of human and Syrian hamster cells

Three approaches have been taken to study simultaneously Syrian hamster cells and human cells in order to develop an extrapolation from the more established hamster system to human cells. On the Characterization of normal cells in comparison to tumor cells, human tumor cells and hamster tumor cells showed similarity in displaying anchorage independence, growth in suspension as micro tumor spheroids, and xenotumorigenicity in contrast to their respective normal cells; in addition, these tumor cells exhibited shorter population doubling time, higher saturation density, higher cloning efficiency, and higher fibrinolytic activity relative to their respective normal cell types. Other differences including ploidy change, contact inhibition on growth, serum requirement, and morphological transformation were also noted between human and hamster cells. On the application of microcarrier culture for a xenotumorigenicity test, the microcarrier technique seemed to have enhanced the sensitivity by reducing the number of inoculated tumor cells required for tumor formation. On the in vitro transformation of normal human and hamster cells, the highest efficiency of morphological transformation of hamster cells has been observed in the group treated with N-methyl-N'-nitro-N-nitrosoguanidine followed by griseofulvin which was employed to enhance the transformation by disturbing the chromosome apparatus. However, no evidence of transformation was observed in the treated human cells thus far.     

Transient gene expression in electroporated Picea glauca protoplasts

The reporter gene for chloramphenicol acetyltransferase (CAT) was introduced into white spruce (Picea glauca (Moench) Voss.) protoplasts by electroporation. CAT transient gene expression was increased by increasing the concentration of pCaMVCN plasmid and was affected by the level of the applied voltage. Highest CAT activities were obtained after electroporation with a pulse of 350V.cm^–1 having an exponential decay constant of approximately 105ms. Linearized plasmid constructs gave much higher levels of CAT activity than circular plasmid. Attempts to use the Escherichia coli -glucuronidase gene (-GUS) as a marker gene revealed very high levels of -GUS-like activity in electroporated protoplasts. This activity was mainly due to a small molecule and may mask successful transformation since -GUS-like activity increased when plasmid DNA was present during electroporation.Abbreviations CAT chloramphenicol acetyltransferase - -GUS -glucuronidase - MUG 4-methyl umbelliferyl glucuronide - F microfarads NRCC No. 29150     

Morphological transformation and catalase activity of syrian hamster embryo cells treated with hepatic peroxisome proliferators,TPA and nickel sulphate

The abilities of the hepatic peroxisome proliferators (HPPs) clofibrate, di(2-ethylhexyl)phthalate (DEHP), mono(2-ethylhexyl)- phthalate (MEHP), 2,4-dichlorophenoxy acetic acid (2,4-D), 2,4,5-trichlorophenoxy acetic acid (2,4,5-T) and tiadenol to induce morphological transformation and to increase the catalase activity of Syrian hamster embryo (SHE) cells were studied. DEHP, MEHP, clofibrate and tiadenol induced morphological transformation of SHE cells and increased the catalase activity. DEHP was more potent than clofibrate and tiadenol in both inducing catalase and morphological transformation, while MEHP seemed more potent than DEHP in inducing catalase, but not morphological transformation, 2,4,5-T and 2,4-D did not induce morphological transformation, but 2,4,5-T was more potent than clofibrate in increasing the catalase activity. These results show that several HPPs induce morphological transformation of SHE cells and an increase in the catalase activity. There is, however, no direct connection between these two parameters, as seen from the results of 2,4,5-T. The tumor promoter TPA, and the metal salt nickel sulphate, induced morphological transformation of SHE cells without any appreciable increase in the catalase activity. These results further corroborate the dissociation between induction of morphological transformation and the increase in catalase activity.Abbreviations Clofibrate ethyl-2-(p-chlorophenox) isobutyrate - 2,4-D 2,4-dichlorophenoxy acetic acid - DEHP di(2-ethylhexyl)phthalate - HPP hepatic peroxisome proliferator - MEHP mono(2-ethylhexyl)phthalate - SHE Syrian hamster embryo - 2,4,5-T 2,4,5-trichlorophenoxy acetic acid - tiadenol di(hydroxyethylthio)-1,10-decane     

Agrobacterium induced gall formation in lipoxygenase mutant isolines of soybeans

Agrobacterium-mediated transformation frequency is very low with cells from some species such as soybeans. Studies were conducted to investigate the Agrobacterium-mediated transformation frequency in near-isogenic lipoxygenase mutant lines of soybeans, since the nigh level of lipoxygenase activity in soybean embryos might be expected to affect interactions with Agrobacterium. The mutant line lacking lipoxygenase 3 showed significantly greater frequency of Agrobacterium-induced transformation than the other soybean lines. Stages of soybean embryo development which showed maximum differences in lipoxygenase 3 activity between mutant and wild-type, also showed maximum differences in transformation frequency. The increased transformation frequency with the absence of lipoxygenase 3 was only seen when both lipoxygenase 1 and 2 were present.Abbreviations Gus -glucuronidase - LB Luria Broth - LOX lipoxygenase - MSO Murashige and Skoog (1962) culture medium with no added hormones - X-GLUC 5-bromo-4-chloro-3-indoyl glucuronide     

Delta5-3beta-hydroxysteroid dehydrogenase and estradiol-17beta-hydroxysteroid dehydrogenase activity in preimplantation hamster embryos

Preimplantation golden hamster (Mesocricetus auratus) embryos were recovered on days 1 (= day of finding spermatozoa in the vagina) through 4 of pregnancy. Postimplantation embryos were studied in sectioned gestation sacs excised on days 5 and 6. Δ⁵-3β-Hydroxysteroid dehydrogenase (3β-HSD) activity in embryos was determined histochemically. There was no enzyme activity on days 1 and 2. Weak activity was first observed at 08:00–09:00 hr on day 3, the activity then increased, peaked at 01:00–03:00 hr on day 4, considerably declined by 08:00–09:00 hr (day 4), and was absent on days 5 and 6. These results suggest that the preimplantation embryos synthesize steroid hormones. It was previously hypothesized (Dickmann and Dey, 1973, Dickmann and Dey, 1974) that, hormones synthesized by the preimplantation rat embryo participate in the regulation of morula to blastocyst transformation and implantation of the blastocyst. This hypothesis is applicable to the hamster.In addition to 3βHSD, estradiol-17β-hydroxysteroid dehydrogenase activity was observed in day 3 embryos, suggesting that the embryo synthesizes estrogen.     

Neoplastic transformation of cultured Syrian hamster embryo cells by DNA of herpes simplex virus type 2.

Syrian hamster embryo cells were transformed to a neoplastic phenotype after exposure to herpes simplex virus type 2 (S-1) DNA at concentrations (less than or equal to 0.01 microgram per 60-mm dish) at which infectivity was no longer demonstrable. Transformed cells manifested in vitro phenotypic properties characteristic of the neoplastic state, expressed herpes simplex virus-specific antigens, and induced invasive tumors in vivo. Transfection and transformation of Syrian hamster embryo cells with herpes simplex virus type 2 DNA or its fragments is a suitable system for investigating the structure and function of herpes simplex virus-transforming gene(s).     

The genetic toxicity of 1,2-dibromo-3-chloropropane, 1,2-dibromo-3- chloro-2-methylpropane,and 1,2,3-tribromo-2-methylpropane

1,2-Dibromo-3-chloro-2-methylpropane (DBCMP) and 1,2,3- tribromo-2-methylpropane (TBMP) are contaminants formed during the manufacture of bromobutyl rubber. These chemicals are structurally similar to 1,2-dibromo-3-chloropropane (DBCP), a known genotoxin and rodent carcinogen. The present study compared the genotoxic properties of DBCMP and TBMP to those of DBCP. In the Salmonella assay, DBCP was positive in strains TA98, TA-100 and TA-1535 in the presence of exogenous activation; DBCP was weakly active in TA-1535 in the absence of activation. Neither DBCMP nor TBMP produced reproducible evidence of mutagenic activity in the Salmonella assay despite the use of several different variations of this test. In the mouse lymphoma gene mutation assay DBCP and TBMP were positive in the presence and absence of activation, while DBCMP was positive only in the absence of activation. All three test compounds were active in the Syrian hamster embryo morphologic transformation assay. The results indicated that both DBCMP and TBMP exhibited some genotoxic activity as did DBCP. The presence of the methyl group on the 2-carbon position essentially eliminated the mutagenicity of DBCMP and TBMP in the Salmonella assay.abbreviations CHO Chinese hamster ovary cells - DBCMP 1,2-dibromo-3-chloro-2-methylpropane - DBCP 1,2-dibromo-3-chloropropane - DMEM Dulbecco's Eagle's minimal E medium - MNNG N-methyl-N'-nitro-N-nitrosoguanidine - S-9 microsomal fraction from rodent liver - TBMP 1,2,3-tribromo-2-methylpropane - TBP 1,2,3-tribromopropane - TFT trifluorothymidine     

Inability of diethylstilbestrol to induce 6-thioguanine-resistant mutants and to inhibit metabolic cooperation of V79 Chinese hamster cells

The mutagenicity of diethylstilbestrol (DES) in V79 Chinese hamster cells was examined under a variety of conditions. DES over a concentration range 0.01–10 μg/ml failed to induce any increase above the spontaneous frequency of 6-thioguanine-resistant V79 cells. The effect of varying the expression time after treatment in the mutation assay from 3 to 9 days was studied and DES was nonmutagenic at all time points, while N-methyl-N′-nitro-N-nitrosoguanidine was highly mutagenic with a peak response after a 5–7 day expression time. The mutagenicity of benzo[a]pyrene and DES, both of which induce morphological and neoplastic transformation of Syrian hamster embryo (SHE) cells, was tested by cocultivating V79 cells with SHE cells for possible metabolic activation of the chemicals. Neither compound was mutagenic to V79 cells in the absence of SHE cells. Benzo[a]pyrene, but not DES, was mutagenic to V79 cells cocultivated with SHE cells. These results support the observation that DES can induce cell transformation under conditions that do not result in any measurable gene mutations. Moreover, the ability of DES to enhance the recovery of 6-thioguanine-resistant mutations was studied by determining the ability of DES to inhibit metabolic cooperation of V79 cells. Unlike the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate, DES was a weak or inactive inhibitor of metabolic cooperation.     

Molecular and cellular mechanisms of transformation of C3H/10T1/2 Cl 8 and diploid human fibroblasts by unique carcinogenic,nonmutagenic metal compounds

Work from our laboratory showed that carcinogenic metal salts of arsenic, nickel, and chromium induced morphological transformation of cultured C3H/10T^1/2 Cl 8 (10T^1/2) mouse embryo cells, and that many of the transformants grow in soft agarose and form tumors in nude mice. Concentrations of arsenic, nickel, and chromium compounds that induced morphological transformation did not induce mutation to ouabain resistance in 10T^1/2 cells. This indicated that the mechanism of metal induced morphological transformation was likely not caused by induction of base substitution mutations, and in the case of lead chromate, likely not caused by frameshift or deletion mutations. In addition, we showed that carcinogenic arsenic, nickel, and chromium compounds, and MNNG, induced anchorage independence in diploid human fibroblasts. Anchorage-independent cell strains derived from anchorage-independent colonies were stable but did not form foci and eventually senesced, therefore, arsenic and nickel compounds and lead chromate induced stable anchorage independence as an isolated phenotype. Nickel compounds and lead chromate induced anchorage independence but not mutation to ouabain resistance or to 6-thioguanine resistance. Hence, the mechanism of induction of anchorage independence by these metal salts in human fibroblasts was likely not via induction of base substitution, frameshift, or deletion mutations that would be measured in these mutation assays. MNNG, on the other hand, induced mutation to 6-thioguanine resistance and to ouabain resistance over the same concentration ranges that induced anchorage independence was induced, indicating that MNNG might induce anchorage independence by inducing base substitution, frameshift, or deletion mutations. It is likely, therefore that the mechanisms of metal salt induced morphological and anchorage independent transformation in murine and human base substitution mutations. We hypothesize that rearrangements or amplification of proto-oncogenes, or, possibly, inactivation of suppressoro oncogenes, might play a role in the mechanism of metal salt induced morphological or anchorage independent transformation of 10T^1/2 and diploid human fibroblasts, respectively. *** DIRECT SUPPORT *** A03GS019 00027     

Receptor activator of NF-κB ligand induces the fusion of mononuclear preosteoclasts into multinucleated osteoclasts

The osteoclasts are bone-resorbing multinucleatedcells formed by the fusion of mononuclearpreosteoclasts (pOCs) of hematopoietic origin.Although receptor activator of NF-Bligand (RANKL) has been shown to regulate osteoclastdifferentiation and function, its effect on the fusionof pOCs into multinucleated osteoclast-like cells(OCLs) has not been known. Using our fusion assaysystem, that is not contaminated with multinucleatedcells (MNCs) and osteoblastic cells, we determined theeffect of RANKL on the fusion of pOCs into MNCs. WhenpOCs were cultured on the plates, most of pOCs diedand disappeared from the plates within 24 h in theabsence of additives, but pOCs were fused to MNCswithin 6 h in the presence of RANKL. RANKL-inducedMNCs showed typical properties of OCL such astartrate-resistant acid phosphatase (TRAP) activity,actin ring formation, and bone-resorbing activity. Thefusion of pOCs into OCLs induced by osteoblastic cellsor RANKL was inhibited by OPG/OCIF, but that inducedby IL-1 was not. Both RANKL- andIL-1-induced OCL formation from pOCs wasinhibited by ZLLL-H, a peptide inhibitor ofproteasome. These findings indicate that RANKLsupports the survival of pOCs and induces the fusionof pOCs into OCLs and suggest that NF-Bactivation is involved in these processes induced byRANKL and IL-1.