Clastogenicity in vitro of the Na, K, Ca and Mg salts of saccharin; and of magnesium chloride; consideration of significance

The sodium, potassium, calcium and magnesium salts of saccharin, and magnesium chloride, have been shown to be clastogenic to Chinese hamster lung (CHL) fibroblasts in vitro, but only at elevated dose levels (8-16 mg/ml). Saccharin acid was inactive to the limits of its solubility (4 mg/ml). When the data are expressed in terms of ionic concentration, each salt showed a similar clastogenic potency. This suggests that ionic effects induced by these salts in the assay medium may be the critical determinant of the clastogenic effects seen, rather than that the saccharin moiety presents a genotoxic insult to the chromosomes of the cells. The metal-chelating agents EDTA and EGTA were non-clastogenic, but the disodium salt of EDTA showed weak activity prior to toxicity at 0.5 mg/ml. The absence of a clastogenic response for the salts of saccharin at dose levels lower than 4 mg/ml is discussed within the context of the threshold-dependent tumour-promoting activity of high dose levels of sodium saccharin to the bladder of male rats. The doubtful value of conducting in vitro clastogenicity studies at dose levels greater than 10(-2) M is discussed.     

The effectiveness of S9 and microsomal mix on activation of cyclophosphamide to induce genotoxicity in human lymphocytes

Comparative results are presented on the effectiveness of rat-liver S9 or microsomal mix (M mix) in activating cyclophosphamide (CP) and its ability to induce a clastogenic effect in human lymphocytes in vitro. Structural chromosome changes were analysed exclusively in 1st division (M1) metaphases post-exposure. A high genotoxic response was observed for both metabolizing systems used. With an exposure of 2 h to different concentrations of S9 or M mix, the highest aberration yields were always found for the highest protein content. For CP treatment times of 1, 2 or 4 h together with S9 mix (protein content 10 mg/ml) or M mix (4 mg/ml), the latter was more efficient. With both systems, a lower clastogenic effect of CP was found at 4 h exposure than at 1 h or 2 h. Only a weak cytotoxic effect, reflected mainly by the reduction in the percentage of 3rd cycle cells (M3), and measured in terms of the proportion of M1, M2 and M3 cells, was induced by both systems.     

Clastogenic action of ellipticine over the cell cycle of human lymphocytes and influence of posttreatments with caffeine and ara-C at G2

The clastogenic potential of the intercalating compound ellipticine, an antitumor alkaloid, has been demonstrated in mammalian cells. To characterize the mechanism of action of this drug over the cell cycle, human lymphocyte cultures from 2 healthy donors were treated with 3 micrograms/ml ellipticine in 30-min pulses during different phases of the cell cycle and analyzed for chromosomal aberrations and sister-chromatid exchanges. The G2 phase was most sensitive in terms of induction of aberrations, followed by S and G1. Chromatid-type aberrations were the most common type of chromosomal damage. Induction of SCEs was significantly high only after treatment at G1, when the frequencies of SCEs doubled. The post-treatment effect of lymphocytes with inhibitors of DNA repair, 10(-3) M caffeine and 5 x 10(-6) M 1-beta-D-arabinofuranosylcytosine, was also tested by adding 3 micrograms/ml ellipticine at G2 in 30-min pulses and immediately followed by caffeine and/or ara-C during the last 3 h before harvesting. Three experiments performed on blood from 3 donors showed a moderate potentiation effect on the frequency of chromatid-type aberrations (about 2-3 times) by both inhibitors. Likewise, a 3-fold increase was observed in the frequencies of chromosomal aberrations when caffeine and ara-C were combined. The present data demonstrate that posttreatment with caffeine and ara-C at G2 can modify the response of human lymphocytes treated with ellipticine by increasing the clastogenic action of this compound or by changing the cell-cycle progression.     

Clastogenic effect of the plant alkaloid ellipticine on bone marrow cells of Wistar rats and on human peripheral blood lymphocytes

Ellipticine (EPC), a natural alkaloid extracted from Aspidosperma williansii (Apocynaceae), is known to have antitumor and cytotoxic activities on various types of tumors. This drug showed a strong clastogenic effect on bone marrow cells of Wistar rats treated in vivo (7.75-31.00 mg/kg body weight). EPC was also tested in vitro using the human peripheral blood lymphocyte system, at concentrations 100 times lower than those used in the in vivo test on rats, since the cytotoxic effect on lymphocytes was very strong. At the 2 highest concentrations used (7.75 X 10(-1) and 1.55 X 10(-1) micrograms/ml culture medium), EPC induced a statistically significant increase in the frequency of chromosome aberrations and sister-chromatid exchanges in lymphocytes. Based on data reported in the literature, we have tried to establish relationships between the clastogenic effect observed and the process of EPC intercalation into DNA and the formation of protein-associated DNA-strand breaks probably promoted by topoisomerase enzymes.     

Tissue-specific clastogenic effects of chromium and selenium salts in vivo

The clastogenic effects of inorganic compounds of chromium (K2Cr2O7) and selenium (Na2SeO3) on the chromosomes of rat lymphocytes and bone marrow have been investigated. In vitro exposure of rat lymphocytes to K2Cr2O7 gave highly significant and dose-related increases in abnormal metaphases at 6 concentrations from 7 X 10(-6) M to 3.2 X 10(-5) M. Similar in vitro exposure of lymphocytes to Na2SeO3 showed that it was clastogenic at concentrations of 7.5 X 10(-6) M, 1 X 10(-5) M and 2.5 X 10(-5) M. However, with in vivo exposures of K2Cr2O7 (i.p. and i.v.) it was only possible to demonstrate clastogenicity in lymphocytes at sublethal concentrations (36 mg/kg X 2 i.v.) and then only if the results were tested against all controls combined (1900 metaphases, 19 animals). On the other hand, very highly significant clastogenic effects were obtained in bone marrow cells exposed in vivo to K2Cr2O7 at 21 mg/kg i.p. and 12, 18, 24 and 36 mg/kg i.v. In vivo exposure to Na2SeO3, with concentrations up to 6 mg/kg X 2 i.v., caused no significant increase in abnormal metaphases in lymphocytes but 5 and 6 mg/kg X 2 i.v. caused a significant increase in abnormal metaphases in bone marrow. These results suggest that K2Cr2O7 and Na2SeO3 are acting as 'S' dependent chemicals. Although not directly comparable, they are compatible with the warnings given by other authors both on the detection of aberrations in lymphocytes after chronic exposure in man and on short-term testing of lymphocytes at low doses related to human exposure.     

Induction of chromosome aberrations by styrene and vinylacetate in cultured human lymphocytes: dependence on erythrocytes

Two vinyl monomers, styrene and vinylacetate, were tested for their ability to induce chromosome aberrations in cultured human lymphocytes. The effects of a 24-h treatment (48 h after culture initiation) were studied both in whole-blood cultures (with 2 X 10(8) erythrocytes/ml) and in isolated lymphocytes (with 4000 erythrocytes/ml). Styrene produced a clear dose-dependent increase in chromatid-type aberrations in whole-blood cultures (0.5-6 mM) and a weaker effect in cultures of isolated lymphocytes (1-4 mM). A statistically significant elevation in aberrations was observed at 2 mM in the former culture type and at 1 mM in the latter. These results support earlier studies on the importance of erythrocytes in the metabolic activation of styrene, but also suggest that a part of this activation occurs in the lymphocytes themselves. Vinylacetate (0.125-2 mM), the more potent clastogen of the two monomers tested, induced a distinct dose-dependent increase in chromatid-type aberrations and a slight elevation in chromosome-type breaks in both culture types. The lowest concentration giving a positive result was 0.25 mM. The clastogenic effects of vinylacetate were somewhat more pronounced in isolated lymphocytes than in whole blood. Vinylacetate is known to be rapidly hydrolyzed in vitro to acetaldehyde, which probably explains the positive result.     

Differences in Escherichia coli Culture Conditions Can Have a Large Impact on the Induction of Extreme Acid Resistance

A recently isolated Escherichia coli strain (3TF4) survived an acid shock that mimicked the low pH of the human gastric stomach (pH 2, 1 h), but this survival was highly influenced by prior growth conditions. Only 0.01% of the stationary phase cells that had been grown anaerobically in a carbonate medium (2 mg glucose and 0.25 mg yeast extract per ml, 40 mm sodium carbonate, final pH 6.5) survived the acid shock, and the survival of exponential phase cultures was even lower (0.0001%). Small amounts of Trypticase (1.5 mg/ml) increased the survival as much as 5000-fold, but cultures that were provided with higher concentrations of Trypticase (7.5 mg/ml) did not reach the stationary phase in 24 h and were more acid sensitive. Sodium acetate (50 mm) also increased acid resistance, and the increased acid shock survival was greater for the cells that had reached the stationary phase (100 versus 1000-fold, respectively). E. coli 3TF4 cultures that had been grown aerobically in Luria broth were already so acid resistant (survivals greater than 40%) that they did not respond to sodium acetate. E. coli 3TF4 cultures that were refrigerated (5°C, 7 days) were nearly as acid resistant as those that were immediately subjected to acid shock (pH 2.0, 1 h). Received: 20 December 2000/Accepted: 7 February 2001     

The effect of ethylnitrosourea on chromosome aberrations in vitro and in vivo

Summary The effect of ENU on (A) human chromosomes from blood lymphocyte cultures in vitro, and on (B) rat and mouse bone marrow chromosomes in vivo, was investigated. Doses of 25, 50, 100 and 200 g/ml were tested in vitro and cells with chromosome breakage were found to be dose dependent. Chromosome damage was also dependent on time; maximum damage was seen when cells were treated 2–6 hrs before harvest.Two doses of 100 and 200 mg/kg were studied in rat and mouse in vivo and a dose effect could be shown in both species. The highest number of abnormal cells was found 6 hrs after treatment; there was a sharp decrease at 18 hrs and thereafter. Types of aberrations were also analyzed, in both in vitro and in vivo studies.     

Combined action of isoniazid and para-aminosalicylic acid in vivo on human chromosomes in lymphocyte cultures

Summary Two antitubercular drugs, viz., isoniazid (INH) and para-aminosalicylic acid (PAS), in combination, were evaluated for their in vivo clastogenic effects on human lymphocyte chromosomes. Lymphocyte cultures from tuberculosis patients taking a therapeutic dose of INH and PAS for a period of not less then 3 months and from two sets of controls were used: (1) newly diagnosed tuberculosis patients who were not yet under therapy and (2) healthy individuals from the general population. Chromosome aberration frequency was very significantly increased in the patients exposed to combined INH and PAS therapy as compared with controls. The most frequently observed aberrations were chromatid breaks and gaps. Isoniazid, the major antituberculosis drug, has been reported not to be clastogenic by itself. However, we observed that the INH-PAS combination commonly used in therapy was clastogenic. From this observation it may be concluded that INH and PAS act synergistically in producing chromosomal aberrations.     

Production of 10-ketostearic acid from oleic acid by a strain of Flavobacterium sp. 12-4A

Summary A new Flavobacterium sp., strain 12-4A, produces solely 10-ketostearic acid (KSA) from oleic acid (OA) in growing cultures. Under optimized conditions 14 mg/ml of KSA was produced from 25 mg/ml of OA after 3 days of cultivation at 30°C.     

Chromosome-damaging action of isoniazid and thiacetazone on human lymphocyte cultures in vivo

Summary Antitubercular drugs in general are given in various combinations, one being isoniazid and thiacetazone. In the present study, was evaluated the in vivo chromosome-damaging effects of a combination of these two drugs in 72 h lymphocyte cultures.Chromosome aberrations were significantly increased in the patients treated with INH and thiacetazone as compared with two types of controls: (1) tuberculosis patients before starting the drug treatment and (2) individuals from the general population. The most frequently observed aberrations were chromatid breaks and gaps.It has been shown that individually, isoniazid may not be clastogenic on human chromosomes in therapeutic doses. The effects of thiacetazone on human chromosomes are not known. Consequently, the enhancement in chromosomal aberrations in the drug-exposed patients may be due to a synergistic effect of isoniazid and thiacetazone or to the clastogenic effects of thiacetazone alone.     

Hydroxynonenal, a component of clastogenic factors?

Exposure of human lymphocyte cultures to superoxide generated by the xanthine-xanthine oxidase (X-XO) system, resulted in formation of a clastogenic factor (CF), as expected from previous work. We speculated that arachidonic acid (AA), the major polyunsaturated fatty acid of biological membranes, was oxidized via the cyclooxygenase-lipoxygenase pathways or nonenzymatically by oxygen free radicals in the culture medium to products with clastogenic properties. In the present study, we analyzed CF for AA-derived products and tested corresponding commercial standards for their clastogenic properties. The results show that prostaglandins, thromboxane, and H(P)ETEs were not increased in supernatants from X-XO treated cultures compared to untreated cultures. Synthetic H(P)ETEs added to the medium of lymphocyte cultures were only slightly or not clastogenic. In contrast hereto, the degradation product 4-hydroxynonenal was found in 50% of CF samples, while it was absent in all 43 control samples. The kinetics of detectability in the culture medium was similar to that of CF. Also, the clastogenic effect of synthetic 4-hydroxynonenal at concentrations as low as 0.1 microM suggested that this aldehyde, known for its genotoxic effects, was a clastogenic component of CF. The indirect action mechanisms of 4-hydroxynonenal via inactivation of functional SH groups in DNA polymerases, may explain why chromatid-type damage is predominant in lymphocytes exposed to CF in the Go-G1 phase of the cell cycle. This particularly was already stressed 20 years ago in the first observations of radiation-induced CF. However, 4-hydroxynonenal is not the only clastogenic component of CF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of pyrimethamine and sulfadiazine on the fine structure and multiplication of Toxoplasma gondii in cell cultures.

Rhesus monkey kidney cell cultures were inoculated with Toxoplasma gondii organisms obtained from peritoneal fluid of mice infected with the RH strain. Pyrimethamine and sulfadiazine were added either singly or in combination to the cultures 4 hr after inoculation. Twenty-four hours later the effect of the drugs on the parasites were studied by light and electron microscopy. Pyrimethamine (1.0 mug/ml) inhibited multiplication of the parasites and caused striking morphological changes. Organisms were rounded and often had a fragmented nucleus. Division was inhibited as indicated by abnormal daughter membrane formation during endodyogeny. No effect was evident in sulfadiazine-treated parasites when concentrations up to 50 mug/ml were used. However, combination of ineffective levels of pyrimethamine (0.1 mug/ml) and sulfadiazine (0.5 mug/ml) produced effects similar to those seen at a higher concentration of pyrimethamine indicating a synergistic action of the 2 drugs.     

Prostacyclin and steroidogenesis in goat ovarian cell types in vitro

Granulosa, theca and corpus luteum cells of the goat ovary were isolated and incubated separately for 6 hours, with or without various modulators. Arachidonic acid (AA, 10 ng to 100 micrograms/ml), the precursor for prostaglandin synthesis, produced a dose-dependent increase in progesterone (P4) and estradiol-17 beta (E2) production by all the cell types. Prostaglandin synthetase inhibitors, aspirin (10(-6)-10(-3)M) and indomethacin (100 ng-1 mg/ml), produced a dose-dependent decrease in arachidonic acid-stimulated (100 micrograms/ml) steroid production. Prostacyclin synthetase stimulators, trapidil (1.6 micrograms- 1 mg/ml) and dipyridamole (10(-6)-10(-3)M), when added alone or along with AA, did not affect steroid production. Up to 100 micrograms/ml of U-51605 (9,11-azoprosta-5,13-dienoic acid), a prostacyclin synthetase inhibitor, did not inhibit basal or AA-stimulated steroid production. Prostacyclin (PGI2) and its stable analog 6 beta PGI1 (0.01-10 micrograms/ml) produced a dose-dependent increase in P4 and E2 production in all the three cell types. Increase at 1 and 10 micrograms/ml was significant in all cases. 6-keto-PGE1 (an active metabolite of PGI2 in certain systems) produced an increase in steroid production which was significant in theca at greater than or equal to 1 microgram/ml concentrations but had no significant effect on granulosa and corpus luteum cells at any dose level. 6-keto-PGF1 alpha (stable metabolite of PGI2) was without effect in the present system. The lack of effect of PGI2 at lower concentrations was not altered by either differentiation of the cells with FSH and testosterone or addition of steroid precursors, testosterone and pregnenolone. The present results indicate that AA-stimulated steroid production in the goat ovarian cell type is mediated by prostaglandins other than PGI2 though PGI2 itself can positively modulate the steroid production.     

Rosmarinic acid production by <Emphasis Type="Italic">Coleus forskohlii</Emphasis>hairy root cultures

Coleus forskohlii hairy root cultures were found to produce forskolin and rosmarinic acid (RA) as the main metabolites. The growth and RA production by C. forskohlii hairy root cultures in various liquid media were examined. The hairy root cultures showed good growth in hormone-free Murashige and Skoog medium containing 3% (w/v) sucrose (MS medium), and Gamborg B5 medium containing 2% (w/v) sucrose (B5 medium). RA yield reached 4.0 mg (MS medium) and 4.4 mg (B5 medium) after 5 weeks of culture in a 100 ml flask containing 20 ml of each medium. Hairy root growth and RA were also investigated after treatment with various concentrations of yeast extract (YE), salicylic acid (SA) and methyl jasmonic acid (MJA). RA production in a 100 ml flask containing 20 ml B5 medium reached 5.4 mg (1.9 times more than control) with treatment of 0.01 or 1% (w/v) YE, 5.5 mg (2.0 times more than control) with treatment of 0.1 mM SA, and the maximum RA content with 9.5 mg per flask (3.4 times more than control) was obtained in the hairy roots treated with 0.1 mM MJA. These results suggest that MJA is an effective elicitor for production of RA in C. forskohlii hairy root cultures.     

Stimulation of citric acid production in Aspergillus niger by addition of viscous substances in shake culture

When glucose (120mg/ml) was used as a carbon source, Aspergillus niger Yang no. 2. showed a markedly low citric acid productivity in shake culture (15.4 mg/ml) but a high productivity in semi-solid and surface cultures (72.3 mg/ml and 67.6 mg/ml, respectively). Since the viscosity of the medium was assumed to be one of the important factors for citric acid productivity in shake culture, the effects of the addition of viscous substances on citric acid productivity of strain Yang no. 2 were examined. The addition of 2.0–6.0 mg gelatin/ml as a viscous additive to the medium containing glucose as a carbon source increased slightly the medium viscosity but substantially increased the citric acid productivity in shake culture to levels of 52.0–53.3 mg/ml, about 3.4 times as much as that without gelatin. However, no influence of gelatin addition was observed in semi-solid and surface cultures, i.e. under static cultivation conditions. Different mycelial morphologies of the strain were observed when cultivations were done in shake culture with or without the addition of gelatin. Addition of 5.0 mg agar/ml, 5.0 mg carageenan/ml, 2.5 mg carboxymethylcellulose/ml and 2.5 mg polyethylene glycol 6000/ml, to the medium containing glucose as a carbon source also increased the citric acid productivity in shake culture to levels of 39.2–54.7 mg/ml. Since Yang no. 2 does not utilize these viscous substances, these results suggested that the viscous substances functioned as protectants for the mycelium from physiological stresses due to shaking and as a consequence resulted in a remarkably increased citric acid productivity in shake culture.     

Embryogenesis in suspension cultures of Datura innoxia Mill.

In vitro plant regeneration via embryogenesis was obtained in suspension cultures of Datura innoxia Mill. Embryogenesis was induced in suspension cultures raised from callus of androgenetic origin, using LS liquid medium supplemented with 0.22 mg/l 2,4-D. The total number of embryos formed was variable over time in culture. Embryos differentiated and matured in the liquid medium itself as also evidenced by histological observation. Embryos germinated to form plantlets on semisolid MS medium without growth substances. The regenerated plants had haploid number of chromosomes.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - Kn Kinetin - LS Linsmaier and Skoog (1965) - MS Murashige and Skoog (1962)     

Effect of hyaluronan on the elastase-type activity of human skin fibroblasts.

The influence of hyaluronan (HA) on the expression of human skin fibroblast elastase-type protease (HSFEp) (Homsy et al, 1988) was studied. At confluency of HSF cultures, hyaluronan increased the level of HSFEp in a time and dose-dependent fashion, Optimal effect was observed after 48 h of culture and at 2 mg/ml HA concentration; the stimulatory, effect of HA could be suppressed by 1 μM cycloheximide. The enhancement of enzyme biosynthesis by HA was dependent on cell proliferation but quasi invariant with HSF passage number (from 7-21).     

Enhanced production of the pharmaceutically important polyphenolic compounds in Vitex agnus castus L. shoot cultures by precursor feeding strategy

Agitated Vitex agnus castus L. shoot cultures were established to analyse the content of selected pharmaceutically important flavonoids and phenolic acids. Two variants (selected from nine ones) of MS medium were prepared: A (BAP 1 mg/L; NAA 0.5 mg/L; GA₃ 0.25 mg/L) and B (BAP 2 mg/L; NAA 0.5 mg/L). The biomass was harvested after 1, 2, 3,4, 5 and 6 weeks. Four‐week cultures (variant A) were selected to perform the precursor feeding experiment. The L‐phenylalanine dose of 1.6 g/L appears to be the most advantageous. Compared to the control cultures, the content of the individual compounds increased in a range from 1.4 to 17.3‐fold (e.g. p‐coumaric acid – 17.3 fold; casticin – 4.8‐fold). The biomass from in vitro cultures is richer in neochlorogenic acid (16‐fold), p‐coumaric acid (5.3‐fold), rutin (2.8‐fold), caffeic acid (1.5‐fold) and cinaroside (1.5‐fold) than the leaves of its parent greenhouse‐cultivated plants. Extracts contained 30 mg/100 g DW of casticin, but after the hydrolysis its amount increased up to 200 mg/100 g DW and twice exceeded the content in the greenhouse leaves. The results indicate that V. agnus castus agitated shoot cultures might be considered as a potential biotechnological source of some pharmaceutically important compounds, especially casticin, rutin, neochlorogenic and p‐coumaric acids.     

Inhibition of human in vitro osteoclastogenesis by Equisetum arvense

Objectives

Equisetum arvense has long been used in traditional medicines to treat different disorders, including bone pathologies. In this study a hydromethanolic extract of E. arvense was assessed for its effects on human osteoclastogenesis.

Materials and methods

Osteoclast precursors were maintained in non‐stimulated and stimulated (presence of M‐CSF and RANKL) conditions, or in co‐cultures with osteoblasts. Cell cultures were treated with 0.00016–0.5 mg/ml of a hydromethanolic E. arvense extract.

Results

The extract did not affect spontaneous osteoclastogenesis. In osteoclast precursors committed to osteoclastogenesis (stimulated or co‐cultured with osteoblasts), E. arvense caused dose‐dependent inhibitory effect that became statistically significant at concentrations ≥0.004 mg/ml. This was observed using different osteoclast differentiation and activation markers. Cell response was associated with changes in relative contribution of MEK and NFkB signalling pathways, as well as PGE2 production. As there were differences in the response of osteoclast precursors maintained in the presence of inductive factors, or co‐cultured with osteoblastic cells, it seems that E. arvense extract had the ability to modulate osteoclastogenesis, either by acting directly on osteoclast precursor cells, and/or via osteoblasts.

Conclusions

Equisetum appeared to have a negative effect on human osteoclastogenesis, which is in line with its putative beneficial role in pathophysiological conditions associated with increased osteoclastic activity, and might suggest potential utility for treatment with bone regeneration strategies.