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A technique using human lymphocytes together with an Ames-type microsomal (S9) activation system for testig indirect chemical mutagens has been developed and examined. The cytotoxic drug cyclophosphamide (CP), which only displays mutagenic properties after metabolic activation, was used as a test chemical and mutagenicity assessed in terms of sister-chromatid exchange (SCE) induction. Direct exposure of lymphocytes to CP and S9 mix produced increases in the yield of SCE for CP concentrations down to 10^?6 M. Exposure times of 30, 60 and 120 min commencing at the beginning of cell culture or after 48 h gave different ranges of detection and response with later treatment being more effective for SCE induction. Variations in relative proportions of the S9 mix culture medium constituents affected the lymphocytes' tolerance of toxic factors and modifiec the mutagen's effect. CP pre-incubated with S9 mix for 1 h before adding to the lymphocyte cultures also produced increases in SCE levels but the method was complicated and did not reduce toxicity. Direct addition of CP and S9 mix to the lymphocytes without prior pre-incubation showed maximum sensitivity, minimum toxocity and was a simpler, more reliable technique.     

Cytogenetic analysis of human peripheral blood lymphocytes in culture exposed in vitro to styrene and styrene oxide

Styrene and styrene oxide mutagenicity was tested in cultured human lymphocytes treated in vitro with various concentrations of test agents. Styrene alone was found mutagenic at the highest concentration used (5 X 10(-4) mol. l-1, combined with the alkylating agent THIO-TEPA it did not affect the chromosome aberration yield. Exposure to styrene oxide gave a positive result showing a clear-cut dose-effect relationship within the concentration range 5 X 10(-6) to 1 X 10(-3) mol. l-1. In combination with THIO-TEPA its effect on chromosome aberration yields was additive. Styrene oxide proved also to be a very potent inducer of sister chromatid exchanges (SCE) within the concentration range 5 X 10(-6) to 1 X 10(-3) mol. l-1 tested. Combined with THIO-TEPA it exhibited a distinct additive effect in the production of SCEs.     

Mutagenicity of some substituted 1-phenyl-3,3-dimethyltriazenes. II. Chromosomal aberrations in human peripheral lymphocytes in vitro

The chromosome-breaking activity of four 1-(phenyl)-3,3-dimethyltriazenes was tested in vitro on human peripheral blood lymphocytes using S9 mix as a metabolic activation system. 1-(4-Nitrophenyl)-3,3-dimethyltriazene was the most active compound. The difference in the frequency of chromosomal aberrations in a test with and without metabolic activation was significant at the 1% level of significance. The lowest frequency of chromosomal aberrations was induced by 1-(4-methylphenyl)-3,3-dimethyltriazene which, under the conditions of this experiment, is the least stable and probably rapidly degraded to non-active compounds. The chromosomal aberrations were also induced by 1-(4-chlorophenyl)-3,3-dimethyltriazene and 1-(4-bromophenyl)-3,3-dimethyltriazene, this activity was unrelated to metabolic activation.     

Mutagenicity of some substituted 1-phenyl-3,3-dimethyltriazenes: II. Chromosomal aberrations in human peripheral lymphocytes in vitro

The chromosome-breaking activity of four 1-(phenyl)-3,3-dimethyltriazenes was tested in vitro on human peripheral blood lymphocytes using S9 mix as a metabolic activation system. 1-(4-Nitrophenyl)-3,3-dimethyltriazene was the most active compound. The difference in the frequency of chromosomal aberrations in a test with and without metabolic activation was significant at the 1% level of significance. The lowest frequency of chromosomal aberrations was induced by 1-(4-methylphenyl)-3,3-dimethyltriazene which, under the conditions of this experiment, is the least stable and probably rapidly degraded to non-active compounds. The chromosomal aberrations were also induced by 1-(4-chlorophenyl)-3,3-dimethyltriazene and 1-(4-bromophenyl)-3,3-dimethyltriazene, this activity was unrelated to metabolic activation.     

Effects of sulfhydryl reagents on Na+-Ca2+ exchange in rat brain microsomal membranes

Effects of six thiol reagents with different physico-chemical properties were tested on the Na+-dependent 45Ca2+ transport into the rat brain microsomal membrane vesicles. The mercurials p-chlormercuribenzoate and Mersalyl effectively inhibited 45Ca2+ uptake with IC50 values in the order of 10(-4) mol X l-1 in the medium. N-ethylmaleimide and its more lipophilic analog N-(4-(2-benzoxazolyl)phenyl)maleimide were much less effective at the same concentrations. 2,2'-dithiodipyridine markedly reduced 45Ca2+ uptake already at concentrations below 10(-4) mol X l-1, whereas 5,5'-dithiobis-2-nitrobenzoate in a concentration range 10(-6)-10(-3) mol X l-1 was a weak inhibitor. Inhibitory effects of the most potent inhibitors p-chlormercuribenzoate and 2,2'-dithiodipyridine were readily reversed by 1 mmol X l-1 dithiothreitol. The results suggest that free SH groups of membrane polypeptides are involved in the functioning of the Na+-Ca2+ exchanger in the nerve tissue cell membranes.     

Clastogenicity of 1-nitropyrene, dinitropyrenes, fluorene and mononitrofluorenes in cultured Chinese hamster cells

The chromosomal aberration test using a Chinese hamster lung cell line (CHL) was carried out on 1-nitropyrene (NP), 3 dinitropyrenes (DNPs), fluorene and 4 mononitrofluorenes with and without metabolic activation (rat S9 mix). The 3 DNPs (1,3-, 1,6- and 1,8-DNP) induced chromosomal aberrations in the absence of S9 mix. The frequencies of cells with aberrations after treatment for 48 h were 43% at 2 micrograms/ml of 1,3-DNP, 55% at 0.1 microgram/ml of 1,6-DNP and 45% at 0.025 microgram/ml of 1,8-DNP, indicating the order of clastogenic potency as 1,8- greater than 1,6- greater than 1,3-DNP. On the other hand, 1-NP, which is known to be a direct-acting mutagen in bacteria, was negative in the chromosomal aberration test without S9 mix, but clearly positive with S9 mix. This effect was dependent on the concentration of the S9 fraction in the reaction mixture. High-pressure liquid chromatography analysis showed that 1-NP was converted by S9 mix to several metabolites, including 1-aminopyrene (AP). The clastogenic activity of 1-AP, however, was equivocal without S9 mix, suggesting that active clastogens other than 1-AP exist. Fluorene induced chromosomal aberrations only in the presence of S9 mix (61.8% at 25 micrograms/ml). 1-, 2-, 3- and 4-nitrofluorene (NF) were more clastogenic in the presence of S9 mix than in the absence of S9 mix, suggesting that NFs were converted to more active clastogens by S9 mix.     

The extrasynaptic effect of gamma-aminobutyric acid and pentobarbital and the influence of temperature on the response of parallel fibres of the frog cerebellum in vitro

The authors studied the effect of two biologically active substances (gamma-aminobutyric acid-GABA, pentobarbital-PB) and a physical factor (temperature-T) on the direct response of parallel fibres of the isolated frog cerebellum to electrical stimulation in vitro. The extrasynaptic action of GABA and PB during superfusion (10(-6), 10(-5), 10(-4) and 10(-3) mol.l-1) significantly reduced the amplitude of the response of parallel fibres. Superfusion with picrotoxin (10(-6) mol.l-1) only partly blocked the effect of GABA (10(-3) mol.l-1), although it abolished the effect of PB (10(-3) mol.l-1). Cooling the cerebellum from the control temperature (T = 16 degrees C) to T = 13 and 10 degrees C significantly augmented the amplitude of the responses, while raising it to 19 and 22 degrees C significantly reduced their amplitude. At T = 13 degrees C, depression of direct responses was significant only in superfusion with GABA (10(-6) and 10(-3) mol.l-1) and not in superfusion with PB (10(-6) and 10(3-) mol.l-1). The results with picrotoxin (PTX) applications, indicated that the extrasynaptic action of GABA and PB took effect by partly different mechanisms. That would account for the difference in the effect of GABA and PB in conjunction with the physical factor.     

Glucose turnover and hormonal changes during insulin-induced hypoglycemia in trained humans

Eight athletes (T), studied the third morning after the last exercise session, and seven sedentary males (C) (maximal O2 consumption 65 +/- 4 vs. 49 +/- 4 (SE) ml X kg-1 X min-1, for T and C men, respectively) had insulin infused until plasma glucose, at an insulin level of 1,600 pmol X l-1, was 1.9 mmol X l-1. Glucose turnover was determined by primed constant rate infusion of 3-[3H]glucose. Basal C-peptide (0.46 +/- 0.04 vs. 0.73 +/- 0.06 pmol X ml-1) and glucagon (4 +/- 0.4 vs. 10 +/- 2 pmol X l-1) were lower (P less than 0.05) and epinephrine higher (0.30 +/- 0.06 vs. 0.09 +/- 0.03 nmol X l-1) in T than in C subjects. During and after insulin infusion production, disappearance and clearance of glucose changed identically in T and C subjects. However, in spite of identical plasma glucose concentrations, epinephrine (7.88 +/- 0.99 vs. 3.97 +/- 0.40 nmol X l-1), growth hormone (97 +/- 17 vs. 64 +/- 6 mU X l-1), and pancreatic polypeptide (361 +/- 84 vs. 180 +/- 29 pmol X l-1) reached higher levels (P less than 0.05) and glucagon (28 +/- 3 vs. 47 +/- 10 pmol X l-1) lower levels in T than in C subjects. Blood pressures changed earlier in athletes during insulin infusion, and early recovery of heart rate, free fatty acid, and glycerol was faster. Responses of norepinephrine, cortisol, C-peptide, and lactate were similar in the two groups. Training radically changes hormonal responses but not glucose kinetics in insulin hypoglycemia.     

Bioenergetics of acclimation to permethrin (NRDC-143) by rainbow trout

Metabolic rates associated with sustained, prolonged and critical swimming speeds were examined in 10 g trout exposed to 5% 96 hr LC50 (0.75 microgram X l-1) and 10% 96 hr LC50 (1.50 micrograms X l-1) at 12 degrees C. Permethrin did not influence the metabolic cost for swimming at sustained and prolonged speeds. Basal metabolic rate increased on initial exposure to permethrin reaching maximum values after 7 days and declined to the control level after 13 days in 5% and after 32 days in 10% 96 hr LC50. Critical swimming speeds were adversely affected in a manner reflective of the effects of permethrin on basal metabolic rate. Elevation in basal metabolic rate in fish exposed to permethrin was a result of increased energy requirements due to physiological stress, detoxication and tissue repair.     

Effects of isoprenaline on tonic tension and Na-Ca exchange in frog atrial fibres

The action of isoprenaline, a purely beta-agonist, was investigated on frog atrial fibres under voltage clamp conditions; tonic tension was induced by long depolarizing pulses and the outward delayed current simultaneously developed. The cumulative dose-response curves showed that isoprenaline increased the peak of tonic tension in the concentration range 10(-8) to 3. 10(-6) mol.l-1, with a maximum effect for 10(-6) mol.l-1. The positive inotropic action of isoprenaline was associated with an increase in the rates of tension rise and of relaxation. Isoprenaline also increased the amplitude of the outward delayed current in a dose-dependent manner. The effects of isoprenaline (10(-6) mol.l-1) on tonic tension and outward delayed current were not observed in the presence of propranolol (10(-7) mol.l-1). Experiments carried out in low-sodium solution demonstrated that the action of isoprenaline on tonic tension can be explained by activation of Na-Ca exchange; the enhanced relaxation might result from the same process. These results suggested that the positive inotropic action of isoprenaline is mediated not only by the well-known increase in the slow inward current but also by activation of the Na-Ca exchange mechanism.     

Influence of S9 mix composition on the SOS response in Escherichia coli PQ37 by polycyclic aromatic hydrocarbons

To investigate the variability in test results obtained with the SOS chromotest (Escherichia coli PQ37 genotoxicity assay) when varying the composition of the exogenous metabolizing system (S9 mix), we examined the influence of different S9 and NADP concentrations, of buffer pH value, of SDS concentrations, the effects of E. coli PQ37 density and centrifugation steps on the expression of β-galactosidase (βg) and alkaline phosphatase (ap) activity, the calculated induction factors (IFs) and SOS-inducing potencies (SOSIPs). Additionally we examined the metabolic potency (stability) of S9 mix when stored at 37°C before use.Initially, we used 0–5000 ng (=0–20 nmole) benzo[a]pyrene (B[a]P) as a reference compound for the test procedure in the presence of standard S9 mix. Subsequently, to evaluate the results of S9 mix variations we examined several polycyclic aromatic hydrocarbons (PAHs) using both the standard and a modified S9 mix composition and test protocol.We observed the highest βg and ap activities and/or IFs using only 11–27 μl 9000 × g liver supernatant (S9) from Aroclor 1254-induced rats per assay (20–50% of standard amount) and calibrating the S9 mix Tris buffer to pH 7.8–8.0. 60–300 μg NADP/assay (10–50% of standard) was sufficient for optimum activation of PAHs. In contrast to previous investigations about the variability of the SOS chromotest in the absence of a metabolizing system, higher induction factors were obtained when using higher bacterial densities (12–18 × 10⁶ cfu/assay). Centrifugation steps as recommended by other investigators were not necessary when using optimum S9 amounts. The metabolic activity of S9 mix remained nearly constant approximately 20 min after preparation, but decreased to 80% of its activity in about 1 h.     

L-asparaginase activity in Aeromonas sp. isolated from freshwater mussel

Aeromonas sp. from Lamellidens marginalis produced L-asparaginase when grown at 37 degrees C. The optimum enzyme activity was at pH 9 when temperature was 45 degrees C. Half-life of partially purified enzyme at 50 degrees C and 55 degrees C was 35 and 20 min, respectively. Activation and deactivation energies of partially purified enzyme were 17.48 and 24.86 kcal mol-1 respectively. The enzyme exhibited a Km (L-asparagine) value of 4.9 x 10(-6) mol l-1 and a Vmax of 9.803 IU ml-1. Three metal ions inhibited the enzyme activity at 10-20 mumol l-1 concentrations. Catalytic activity was also inhibited by EDTA, iodoacetic acid, parachloromercuribenzoic acid and phenylmethylsulphonyl fluoride at 0.1 mumol l-1.     

Analysis of cytogenetic effect in human lymphocytes induced by metabolically activated 2-nitropropane

Chromosome analyses were carried out in human lymphocytes treated in vitro with 2-nitropropane (2-NP) in the presence and absence of the mammalian metabolic activation system, S9 mix. Without S9 mix, only the frequency of gaps was significantly increased at 80 mM 2-NP as compared to controls. With S9 mix, the incidences of gaps and chromatid-type aberrations were significantly increased at 60 mM and 80 mM. Sister-chromatid exchanges (SCE) have been induced at concentrations as low as 7.5 mM. The present findings demonstrate that in human lymphocytes, 2-NP requires metabolic activation to express clastogenicity and SCEs.     

Chromosomal aberrations induced in vitro by 3,7- and 3,9-dinitrofluoranthene

The chromosomal aberration test using a Chinese hamster cell line (CHL) was carried out with 3,7- and 3,9-dinitrofluoranthene (DNF) with and without exogenous metabolic activation (rat liver S9 mix). The highest dose tested was limited to 20 μg/ml because of the compounds' insolubility in dimethyl sulfoxide. Both DNFs induced chromosomal aberrations in the absence of S9 mix; the frequency was not very high. Results were reproducible, but without clear dose-response relationships. Neither DNF induced chromosomal aberrations in the presence of S9 mix. Both DNFs did not induce polyploid cells under any conditions.     

VIP receptors in pig liver cells: binding characteristics and role in degradation

The physiological role of VIP in the liver is controversial. VIP receptors are present, but their function in the metabolic regulation is uncertain. The interaction of porcine VIP with isolated cells from pig liver was studied with respect to receptor-binding, degradation and glycogenolytic action. In this model, VIP and liver showed homology of animal species. 1. Receptor-binding was heterogenous with Kd values of 10(-9) mol/l and 4 X 10(-8) mol/l, and a total amount of binding sites of 7 X 10(-11) mol per 10(9) cells. The peptide specificity showed that porcine and chicken VIP were equally potent in inhibiting receptor-bound 125I-VIP; secretin was about 30 times less potent; glucagon and somatostatin were ineffective. 2. Receptor-bound 125I-VIP was degraded since about 70% was released as radioactivity not reacting with VIP-antiserum. 3. Glucose-release was not stimulated by VIP (10(-6) mol/l) whereas the rate was increased two-fold by glucagon (10(-6) mol/l). In conclusion, VIP receptors in pig liver cells are different from other tissues regarding peptide specificity. It is suggested that receptor-binding mediates degradation of VIP by pig liver rather than metabolic effects.     

Reverse mutation tests in Salmonella typhimurium and chromosomal aberration tests in mammalian cells in culture on fluorinated pyrimidine derivatives

Reverse mutation (Ames) tests with Salmonella typhimurium TA98, TA100 and TA1537, and chromosomal aberration tests in vitro with a Chinese hamster fibroblast cell line (CHL), were carried out on fluorinated pyrimidine derivatives, such as 5-fluorouracil (5-FU), 1-(2-tetrahydrofuryl)-5-fluorouracil (FT), 5-fluorodeoxyuridine (FUdR), 1,3-bis(2-tetrahydrofuryl)-5-fluorouracil (FD-1) and a mixture of uracil and FT in the molar ratio 4 : 1 (UFT) (Fujii et al., 1978). For comparison, similar tests were also carried out on 4 anti-metabolic agents, a metabolite of FD-1 and a component of UFT, such as cytosine-1-beta-D-arabinofuranoside (AraC), 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), 8-azaguanine (8-AG), 3-(2-tetrahydrofuryl)-5-fluorouracil (3-FT) and uracil. The anti-bacterial action of 4 fluorinated pyrimidine derivatives such as 5-FU, FT, FD-1 and UFT to TA100 was tested under the condition that buffer, S9 mix, S9 and albumin were present. 6-MP was only positive in the Ames test with TA100 in the system without S9 mix, while all others failed to show mutagenic activity. On the other hand, all compounds tested, except uracil, induced chromosomal aberrations on CHL cells in the system without metabolic activation. FT was degraded by S9, but there was no significant difference in the killing activity of FT among with buffer, S9 mix and albumin. The killing activity of 5-FU was the strongest with buffer, and it was slightly binding to albumin. The killing activity of 5-FU was mostly decreased by S9 mix. FD-1 showed the strongest anti-bacterial action when S9 mix was present but it was degraded by S9. UFT showed no anti-bacterial action in any conditions.     

Mutagenicity of K-region derivatives of 1-nitropyrene; remarkable activity of 1- and 3-nitro-5H-phenanthro[4,5-bcd]pyran-5-one

The mutagenic activities toward S. typhimurium strains TA98 and TA100 of K-region derivatives of 1-nitropyrene and pyrene were determined. The compounds tested were trans-4,5-dihydro-4,5-dihydroxy-1-nitropyrene (Compound 3), trans-4,5-dihydro-4,5-dihydroxypyrene (Compound 4), 1-nitropyrene-4,5-quinone (Compound 5), 1-nitropyrene-9,10-quinone (Compound 6), pyrene-4,5-quinone (Compound 7), and the lactones, 1-nitro-5H-phenanthro[4,5-bcd]pyran-5-one (Compound 8), 3-nitro-5H-phenanthro[4,5-bcd]pyran-5-one (Compound 9), and 5H-phenanthro[4,5-bcd]pyran-5-one (Compound 10). Neither pyrene nor any of its K-region derivatives was mutagenic, either in the absence or presence of S9 mix at the doses tested. Of the K-region derivatives of 1-nitropyrene, the lactones (Compounds 8 and 9) were generally the most active; 0.25 microgram/plate induced 900-2200 revertants in TA98 or TA100 without activation. The 4,5-dihydrodiol (Compound 3), an established mammalian metabolite of 1-nitropyrene, was less mutagenic than was 1-nitropyrene in TA98, but was more mutagenic than was 1-nitropyrene in TA100, regardless of the presence of S9 mix. The quinones (Compounds 5 and 6) were less mutagenic than was 1-nitropyrene in the absence of S9 mix in both strains, but their activities were increased in the presence of S9 mix. The mutagenic activities of the lactones (Compounds 8 and 9) were lower in strains TA98NR and TA98/1,8-DNP6 than in TA98, indicating that nitro-reduction and esterification are involved in their activation. The results of this study indicate that K-region derivatives of 1-nitropyrene may be important in its metabolic activation.     

Effect of pH shifts on the mutant frequency at the thymidine kinase locus in mouse lymphoma L5178Y TK+/- cells

Evidence has been accumulating that conditions of nonphysiological pH may affect the results of in vitro genetic tests by mechanisms unrelated to the chemical being tested. Medium was pH-adjusted with HCl, NaOH or with organic buffers (Good's zwitterions). In the absence of S9 mix, no changes in mutant frequency were observed over a pH range of 6.4-9.2; a small, 1.9-fold increase was observed for a moderately toxic treatment (24% relative growth) at pH 6.3. However, in the presence of S9 mix, the mutant frequency increased sharply for pH values below 6.8. At pH 6.4, a 4-fold increase was induced, and pH 6.0 resulted in a 10-fold increase in mutant frequency. Basic pH shifts in the presence of S9 mix caused no changes in mutant frequency up to pH 8.0; treatment with pH 8.8 was highly toxic (5.3% relative growth) and caused a 3-fold increase in mutant frequency. Thirteen mutant clones induced at pH 6.0 with S9 mix were challenged with trifluorothymidine after their expansion in nonselective medium and all retained their resistance; another 14 clones were tested for thymidine utilization and all incorporated only 0.1-5.5% of the 14C-labeled thymidine used by the parental line. The induced mutants were primarily of the small-colony phenotype, which indicated clastogenic activity. This was confirmed with chromosome studies which showed a large increase in cells with aberrations consisting of chromatid breaks and complex rearrangements. The results show that the combination of weak acidity (pH 6-6.8) and S9 mix is mutagenic and clastogenic to L5178Y TK+/- cells.     

Chromosomal aberration tests on 29 chemicals combined with S9 mix in vitro.

A metabolic activation system with rat-liver microsome fraction plus cofactors (S9 mix) was applied to chromosomal aberration tests in vitro for the screening of chemical mutagens or carcinogens in the environment. Dialkylnitrosamines only induced chromosomal aberrations in Chinese hamster cells (CHL) when treated with S9 mix. The incidence of chromosomal aberrations in CHL varied with experimental conditions, e.g. incubation time, recovery time, components of S9 mic and inducers used for preparation of S9. For dimethylnitrosamine (DMN), the maximal incidence was obtained when the cells were incubated with S9 mix for 3 h and harvested 24 h after treatment. Therefore, this system (3 h incubation and 24 h recovery) was routinely applied to further screening of other chemicals with S9 prepared from PCB-pretreated rats. 10 carcinogens (e.g. 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene, quinoline, etc.) out of 16 induced aberrations when they were treated with S9 mix, whereas the remaining 6 carcinogens (e.g., 3-methyl-cholanthrene, 4-o-tolylazo-o-toluidine, etc.) induced few or no aberrations even after activation. Two insecticides, allethrin and diazinon, were strongly positive at relatively low doses only when they were activated with the S9 mix. Medical drugs, such as ethenzamide, methyl p-hydroxybenzoate and nitrofurazone, and a food additive, sodium hypochlorite, were positive on activation. Chemicals used for industry, such as styrene monomer and tris-dichloropropylphosphate, were also positive in our activation system.     

Hypersensitive character of Bloom syndrome B-lymphoblastoid cell lines usable for sensitive carcinogen detection

Various carcinogens were tested with regard to the induction of sister-chromatid exchanges (SCEs) and chromosome aberrations using 3 types of Bloom syndrome (BS) B-lymphoblastoid cell lines (LCLs) (type I with normal frequency of SCEs and normal karyotype; type II with high frequency of SCEs and normal karyotype; type III with high frequency of SCEs and abnormal karyotypes) in the presence and absence of S9 mix. Three types of BS B-LCLs and normal cells showed different responses to the various carcinogens in the level of SCE induction. BS type I cells had the same SCE response as normal cells to carcinogens. Some carcinogens that require metabolic activation (S9 mix) had little effect on type II cells without S9 mix but had high SCE levels with S9 mix. BS type III cells were highly susceptible to both direct and indirect carcinogens with respect to high SCE increase without S9 mix (ca. 140 SCEs/cell), though some carcinogens produced SCEs rated in the medium (ca. 120 SCEs/cell) range, and had a high rate (more than 10%) of centromere spreading (CS), in addition to quadriradials. Therefore BS type III is a unique cell line which can be used to detect carcinogens.