The action of anticlastogens in human lymphocyte cultures and their modification by rat-liver S9 mix. II. Studies with vitamins C and E

The action of vitamins C (VC) and E (VE) on the clastogenic activity of trenimon (TR), cyclophosphamide (CP) and bleomycin (BM) was tested on cultures of human peripheral blood lymphocytes with and without addition of rat-liver S9 mix. In addition, the influence of both anticlastogens on the SCE-inducing activity of TR and CP was examined under the same conditions. A distinct dose-dependent anticlastogenic effect of VC was detected in the action of long-term treatment (24 h) with TR, if the vitamin was added to the cultures simultaneously with or before the clastogen. In the short-term tests (2 or 3 h clastogen treatment ending 23 h or 21 h before harvesting) simultaneous addition of both vitamins did reduce the chromosome-damaging action of TR whether S9 mix was present or absent. While VC also decreased the frequency of chromosome damage induced by S9-mix-activated CP, VE was inactive under the same conditions. Neither vitamin significantly affected the chromosome-breaking activity of BM if S9 mix was absent, but they increased the clastogenicity of BM metabolized by S9 mix. In contrast to their anticlastogenic efficacy neither of the vitamins displayed any significant anti-SCE effect, nor were they active in affecting the inhibition of cell proliferation caused by TR or CP.     

The anticlastogenic effect of various combinations of cysteamine,AET, HCT,and amino acids on chromosome damage by Trenimon and bleomycin in human lymphocytes in vitro

Summary The protective activity of the combined application of anticlastogens against the chromosome-damaging action of Trenimon and bleomycin was studied by analyzing more than 32000 metaphases from cultures of human peripheral lymphocytes. Screening tests with the combinations cysteine/cysteamine/AET, cysteine/methionine/asparagine, cysteine/serine/HCT, and AET/HCT, with Trenimon as clastogen, in no case revealed a hyperadditive (synergistic) effect. From the results of detailed analyses of the action of the combination AET/HCT it was concluded that the (nonsynergistic) anticlastogenic effects observed were induced due to intracellular biologic mechanism, and not due to a reaction in the culture fluid between clastogens and anticlastogens. Although the observations gained with Trenimon or bleomycin differed in some respects, the anticlastogens apparently act via a mechanism at least common to AET and HCT, i.e., they manifest their effect in lymphocyte cultures by a limited interaction with the process of aberration formation, rather than by influencing repair processes, which are blocked by caffeine.Dedicated to Professor Dr. med. Gerhard Koch on the occasion of his 65th birthday     

Comparative studies on the specificity of anticlastogenic action in human lymphocytes in culture

Summary Comparative studies on human lymphocyte cultures yielded a certain specificity of the anticlastogenic action of the SH compounds 1-cysteine, cysteamine, and -aminoethylisothiouronium (AET) as well as of the amide 1-asparagine and the amino acid 1-methionine. This specific anticlastogenic activity manifested itself in specific changes of the spectrum of aberration types induced by the clastogens and of the pattern of intercellular distribution of the induced aberrations. It was clearly dependent on the concentration of the anticlastogens but was also influenced by the used clastogen. The use of different culture media yielded some quantitative influences on the anticlastogenic activity, but fundamental changes in the spectrum of anticlastogenic action have not been observed except with cysteamine. The patterns of activity ascertained for the different anticlastogens specifically differed from those changes in the spectrum and pattern of distribution of aberrations induced by a mere reduction of the concentration for instance of Trenimon. Therefore a direct reaction between the protectors and the clastogen Trenimon as the cause of the observed anticlastogenic action was again excluded. The presented data are also discussed under the aspects of the hypotheses of aberration induction as well as of their importance for further antimutagen research.Some parts of this paper have been supported by grants of Deutsche Forschungsgemeinschaft.     

Anticlastogenic effects of black tea (World blend) and its two active polyphenols theaflavins and thearubigins in vivo in Swiss albino mice.

This study investigated the inhibition of cyclophosphamide (CP) and dimethylbenz(a)anthracene (DMBA) induced genetic damage by black tea (World blend) and its two active polyphenols theaflavins (TF) and thearubigins (TR) in Swiss albino mice as measured by chromosome aberrations (CA) and sister chromatid exchanges (SCE). Three different concentrations (5, 10 and 20%) of tea and a single dose of TF and TR were tested for their anticlastogenic effects against DMBA (50 mg/kg body weight) and CP (20 mg/kg for CA and 10 mg/kg for SCE). A significant decrease in CA was observed in all the three concentrations of tea extract plus DMBA treated groups when compared with the respective DMBA treated group alone. Similarly a significant decrease in CA was observed in all the three concentrations of tea extracts plus CP treated series when compared with the group treated with CP alone. In SCE assay, a significant decrease in SCE was observed in 5, 10 and 20% black tea extract plus CP and 10 and 20% tea extracts plus DMBA treated groups when compared with the CP or DMBA treated group alone. In the single dose of TF and TR treated groups a significant decrease in both CA and SCE was observed in both the TF and TR plus both the carcinogen treated groups when compared with their positive controls. The protective effects of black tea extracts were more significant than that of its two polyphenols. This study indicates that both black tea and its active polyphenols TF and TR have significant anticlastogenic effects in bone marrow cells of mice.     

The activity of bleomycin and modifiers of its clastogenic effect on the interphase chromatin of indian muntjak fibroblasts

Premature chromosome condensation was induced in Indian muntjak fibroblasts after exposure of the cells to bleomycin. Further experiments were devoted to the interaction of anticlastogens and a repair inhibitor, streptovitacin A. Chromosomal aberrations due to bleomycin treatment were S -phase-independently visible in the GI and G2 phase of the cell cycle. For premature chromosome condensation experiments, a 100 fold lower concentration of the mutagen produced a similar extent of chromosome damage as in metaphase studies. Additional exposure to the anticlastogens -aminoethylisothiouronium or N-acetylcysteine revealed differences between corresponding interphase and metaphase effects and between different exposure conditions. Streptovitacin A, known as an inhibitor of protein synthesis, acted like an anticlastogen in the G2 phase of the cell cycle. Our studies show that the premature chromosome condensation technique offers various qualitative insights into primary processes of mutagenicity and antimutagenicity, but requires further improvement and careful choice of the cell system for study.Abbreviationd AET -aminoethylisothiouronium - BM bleomycin - CHO Chinese hamster ovary - DMSO dimethylsulfoxide - FBS fetal bovine serum - NAC N-acetylcysteine - PCC premature chromosome condensation prematurely condensed chromosomes - PEG polyethylene glycol - SA streptovitacin A     

Genotoxic effects of fluoride evaluated by sister-chromatid exchange

The purpose of this investigation was to study the genotoxic potential of fluoride (in the form of sodium fluoride, NaF) using in vitro and in vivo sister-chromatid exchange (SCE) assays with Chinese hamster cells. The NaF concentrations used in cultures of Chinese hamster ovary (CHO) cells ranged from 0 to 6.3 mM, both with and without S9 activation. Fluoride analysis of the culture medium demonstrated that it contained little indigenous fluoride, and the concentration of added fluoride was not affected by the components of the medium or the S9 mix. The CHO cells cultured in 6.3 mM NaF almost vanished, and at the concentration of 5.3 mM NaF in cultures without S9 microsome, only M1 cells were observed. In in vivo studies, Chinese hamsters were intubated with NaF dosages of 0, 0.1, 1.0, 10, 60 and 130 mg/kg, and the bone marrow (CHBM) cells were examined for SCE frequencies. Bone fluoride data showed that the intubated NaF was effectively absorbed. Death occurred in 3 of the 8 animals given 130 mg NaF/kg. The results indicated that NaF, in dosages up to 5.3 mM in CHO cell cultures and 130 mg/kg in in vivo CHBM cells, did not significantly increase the SCE frequencies over those observed in the negative (distilled water) controls. However, examination of the cell cycle revealed an inhibitory effect of NaF on cell proliferation with doses of NaF at or greater than 1.0 mM in cultured CHO cells and at or greater than 60 mg NaF/kg in in vivo CHMB cells. The results of the present study indicated an inhibition of the cell cycle and death of the cells with increasing concentrations of fluoride but not effect of fluoride on SCE frequency in CHO and CHBM cells.     

Strong antimutagenic effects of fluoride on mutation induction by trenimon and 1-phenyl-3,3-dimethyltriazene in Drosophila melanogaster

After fluoride treatment of mature and immature oocytes of Drosophila females, a clear-cut dose-dependent decrease in fertility and fecundity was observed. The hatchability of mature oocytes was reduced by as much as 35%. When immature oocytes were treated, a pronounced dose-dependent reduction in fecundity occurred.

Exposure of mature sperm to NaF resulted in a slight decrease in fertility, comparable to the effect obtained with immature oocytes. Of the criteria used to measure possible mutagenic effects of NaF (sex-linked lethals, partial and total X- and Y-chromosome losses), only the rate of total losses was enhanced significantly.

The slight mutagenic effect of NaF on mature sperm was not related to the strong antimutagenic activity observed, when applied simultaneously with any of the several chemical mutagens. NaF treatment drastically reduced both the Trenimon-induced decrease in fertility and Trenimon-induced increases in recessive lethal mutation frequencies and rates of partial and total chromosome losses. The inhibitory effect of NaF was less pronounced with 1-phenyl-3,3-dimethyltriazene (PDT), a poor chromosome breaker in Drosophila, and absent for A 137, a weak mutagen which so far has failed to induce chromosomal aberrations in Drosophila. Therefore, the data are interpreted as being the result of a specific fluoride inhibition of chemically induced chromosomal breakage.

In mature and immature oocytes, the decreases in fertility and fecundity, and increase in recessive lethal frequency (mature oocytes) produced by Trenimon were also suppressed in the presence of fluoride. However, since Trenimon failed to produce a significant rise in X losses and NDJ in both stages, the effect of NaF on these mutational classes was, of course, not testable.     

Induction of sister-chromatid exchanges in human lymphocytes and Chinese hamster cells exposed to aflatoxin B1 and N-methyl-N-nitrosourea.

The effect of a 1-h exposure to aflatoxin B1 (AFB1) in inducing sister-chromatid exchange in Chinese hamster ovary (CHO) cells and human lymphocytes in the presence or absence of mixed function oxidase ("S9 mix") was compared. CHO cells were also exposed to a graded series of doses of N-methyl-N-nitrosourea, a powerful inducer of SCE whose action was independent of the presence or absence of S9 mix. CHO and human cells showed a close correlation in response to SCE induction by AFB1 and in both cell systems the additon of mixed function oxidases in the S9 mix resulted in a marked enhancement of action of AFB1.     

The genotoxic and cytotoxic effects of ribavirin in rat bone marrow

The genotoxicity of the 2-furylethylene derivative 1-(5-bromofur-2-yl)-2-nitroethene (2-betaNF) has been evaluated in cultured human peripheral blood lymphocytes at concentrations ranging from 0.5 to 15microg/ml. The frequencies of micronuclei (MN) and sister-chromatid exchanges (SCEs) were used and scored as indicators of genetic damage. To asses the role of the metabolism mediated by the enzymes present in the S9 mix, over the possible genotoxic potential of the test agent, the cultures for MN and SCE demonstrations were treated for 3h in presence and in absence of rat liver microsomal fraction. The results indicate that, under the experimental conditions used, the test agent does not induce significant increases in the frequency of micronucleated cells, irrespective of the presence/absence of metabolic fraction. Nevertheless, a slight increase in the SCE frequency was observed in those cultures treated without the S9 mix; although this slight increase disappeared in the experiments carried out with the microsomal fraction. In addition, cytotoxic/cytostatic effects of (2-betaNF) were observed mainly in the cultures treated without the S9 fraction.     

The rate of sister chromatid exchanges parallel to spontaneous chromosome breakage in Fanconi's anemia and to trenimon-induced aberrations in human lymphocytes and fibroblasts.

The incidence of structural chromosome aberrations and the rate of sister chromatid exchanges (SCE) was investigated in lymphocyte cultures from a patient with typical Fanconi's anemia and his parents. The rate of SCEs was found to be normal. In experiments with the alkylating agent Trenimon the SCE rates proved to be a sensitive indicator for the induction of structural aberrations: in presence of an induced aberration rate half as high as the spontaneous rate in the Fanconi's anemia case, the rate of SCEs was found to be quintupled. Dose-effect relationships for the induction of SCE rates by Trenimon were studied over a wide dose range in lymphocyte and fibroblast cultures. The results reflect the same difference in sensitivity earlier observed in the induction of structural chromosome aberrations, fibroblasts being far more sensitive.     

Effect of tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate on induction of sister-chromatid exchanges in Chinese hamster V79 cells treated with mutagens

The effect of a tumor promoter, 12-O-tetradecanoyl-phorbol 13-acetate (TPA) alone and in combination with mitomycin C (MMC) or cyclophosphamide (CPP) on the induction of sister-chromatid exchanges (SCE) in Chinese hamster V79 cells was investigated. TPA alone at various doses and durations caused no increase of SCE frequency. MMC either at the dose of 0.03 or 0.003 μg/ml alone or in combination with TPA (2 μg/ml) all caused a significant increase of SCE frequencies. There was no difference in SCE frequencies between the cultures treated with MMC alone at 0.03 μg/ml and those treated with MMC plus TPA. However, cultures treated with MMC at 0.003 μg/ml plus TPA had significantly and consistently higher SCE frequencies than those treated with MMC alone at all durations. Treatment of CPP at 1 μg/ml activated by S9 mix caused significant increase of SCE frequencies. Surprisingly, the cultures treated with CPP, S9 mix plus TPA (2 μg/ml) caused a drastic reduction of SCE frequencies as compared to those treated with CPP and S9 mix only at all durations. These results indicate that TPA alone had no effect on SCE in V79 cells. TPA enhanced the SCE induction in V79 cells treated with MMC at a low dose, i.e. 0.003 μg/ml, but it inhibited SCE induction in cultures treated with the indirect mutagen CPP. Thus, TPA has no direct effect on genetic materials but it may indirectly alter the effects of a mutagen.     

Author index

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.     

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.     

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.     

Tea tannin components modify the induction of sister-chromatid exchanges and chromosome aberrations in mutagen-treated cultured mammalian cells and mice

The modifying effects of tannin components extracted from green tea and black tea on mutagen-induced SCEs and chromosome aberrations were studied. These tannin components did not affect spontaneous SCEs and chromosome aberrations in cultured Chinese hamster cells. The frequency of SCEs and chromosome aberrations induced by mitomycin C (MMC) or UV was enhanced by the posttreatment with tea tannin components. When cells were post-treated with tea tannin components in the presence of metabolic enzymes of rat liver (S9 mix), the modifying effects on the induction of SCEs and chromosome aberrations by mutagens were complicated. MMC- and UV-induced SCEs and chromosome aberrations were suppressed by the posttreatment with tea tannin components at low concentrations (less than or equal to 6.7 micrograms/ml) with S9 mix. At a high concentration of tea tannin components (20 micrograms/ml) with S9 mix, a co-mutagenic effect was observed. The modifying effects of tea tannin components were shown to occur in the G1 phase of the cell cycle. In cells from a patient with xeroderma pigmentosum (XP) and a normal human embryo, MMC-induced SCEs were suppressed by the posttreatment with tea tannin components in the presence of S9 mix, and enhanced in the absence of S9 mix. On the other hand, tea tannin components modified SCE frequencies in UV-irradiated normal human cells but not in UV-irradiated XP cells. Our results suggested that tea tannin components themselves inhibited DNA-excision repair and resulted in a co-mutagenic effect, while in the presence of S9 mix metabolites of tea tannin components promoted DNA-excision repair activity and resulted in an antimutagenic effect. MMC-induced chromosome aberrations in mouse bone marrow cells were suppressed by the pretreatment with green tea and black tea tannin mixture.     

Differences in the induction of SCEs between human whole blood cultures and purified lymphocyte cultures and the effect of an S9 mix

Studies for SCE induction are frequently performed on human blood cultures. Either whole blood cultures (WBC) or purified lymphocyte cultures (PLC) are employed. However, it has been shown that fundamental differences with respect to metabolic activity exist between these two systems. In order to further characterize the whole blood culture and the purified lymphocyte culture, differently acting substances were studied comparatively with and without an Aroclor-1254-induced S9 mix. Treatment with ethyl methanesulfonate (EMS), a direct mutagen, produced distinct SCE induction in both systems. Cyclophosphamide (CP) and benzo[a]pyrene (BP), two indirect mutagens, also led to a significant increase of SCEs both in WBC and PLC without S9 mix. Only with CP was this effect more pronounced after addition of S9 mix. Sodium selenite (Na2SeO3), which induced SCEs in WBC, did not show this effect in the PLC. After S9 mix was added to purified lymphocytes, an increase of SCEs by sodium selenite was observed as in WBC. H2O2, a radical former, led to SCE induction in purified lymphocytes but not in the whole blood culture. By adding S9 mix, a distinct reduction of the SCEs induced by H2O2 was established. These results show that human lymphocytes can metabolize indirect mutagens and that it should be kept in mind when using S9 mix that, besides mixed-function oxygenases, it also contains enzymes which influence the SCE-inducing effects of substances.     

The clastogenic effect of 5-methoxypsoralen plus UV-A in human lymphocytes in vitro and its modification by the anticlastogen β-aminoethylisothiouronium

Summary The treatment of human lymphocytes in vitro with 5-methoxypsoralen (5-MOP) plus UV-A induces a dose-dependent increase in the SCE rate and in structural chromosome aberrations. We carried out tests to see whether the clastogenic effect of 5-MOP plus UV-A could be reduced by the anticlastogen -aminoethylisothiouronium (AET). The occurrence of a protective effect proved to be dependent upon the conditions of treatment. When AET was present over a long period of time (22h) in cultures with 5-MOP, the number of breaks was reduced compared with such cultures without AET (reduction factor 0.5–0.6). On the other hand, a short period of action by AET (1.5 h) in the presence of 5-MOP produced no reduction of breaks. Posttreatment with AET (20 h) yielded an obvious protective effect (reduction factor 0.2–0.4). The possible mechanisms of the protective effect of AET are discussed.     

The possible role of probiotics as dietary antimutagen.

Possible antimutagenic actions of probiotics--mainly lactic acid bacteria--were examined using in vitro and in vivo test systems. In the Ames test with Salmonella typhimurium TA1538 beef extract and nitrosated beef extract were used as mutagens. L. casei showed high antimutagenic activity on mutagenicity induced by nitrosated beef extract only without S9 mix, whereas Omniflora (a lyophilized preparation of lactobacilli and E. coli) and its cell-free culture broth exhibited antimutagenic action only on beef extract. The actions of probiotics were more homogeneous when living animals were used in the tests. Using busulfan as a mutagen both the chromosome aberration test (with Chinese hamster bone marrow cells) and the micronucleus test (with bone marrow cells of Chinese hamsters and mice) showed strong anticlastogenic action when L. casei, Omniflora or yoghurt (with living bifiobacteria) were given orally at the same time as the mutagen. Lactobacilli were effective also after i.p. injection. Cell-free culture broths had no or only weak antimutagenic effects. Mutagen-induced chromosome aberrations and micronuclei were reduced by up to 80% by the lactobacilli.     

Mechanism of anticlastogenicity of Agaricus blazei Murill mushroom organic extracts in wild type CHO (K(1)) and repair deficient (xrs5) cells by chromosome aberration and sister chromatid exchange assays

Agaricus blazei Murill is a medicinal mushroom native to Brazil. The present work assessed the clastogenic and anticlastogenic potential of organic extracts (ethanol and chloroform/methanol) from the lineage AB97/11 in chinese hamster CHO-K(1) (wild type) and CHO-xrs5 (repair deficient) cells using the chromosome aberration (CA) and sister chromatid exchange (SCE) assays. In these experimental conditions were observed: (a) anticlastogenic effect at concentrations of 0.06 and 0.09% of the EtOH extract and at the 0.03 and 0.06% concentrations of the C/MetOH extract in CHO-K(1); (b) absence of protector effect on CHO-xrs5 cells; and (c) absence of protector effect in the SCE assay. These results indicate that organic extracts of A. blazei lineage AB97/11 present bio-antimutagenic type protective activity.     

Genotoxic effects of chemicals in the single cell gel (SCG) test with human blood cells in relation to the induction of sister-chromatid exchanges (SCE)

In a comparative study, henzo[a]pyrene (BaP), cyclophosphamide (CP), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and tetrachloroethylene (PER) were tested for their ability to induce genotoxic effects in the single cell gel (SCG) test and the sister-chromatid exchange (SCE) test with human blood cells. MNNG as well as S9 mix activated BaP- and CP-induced DNA effects in both tests in a dose-dependent manner. While the range of concentrations which induced DNA migration or SCE was the same for MNNG and for Bap, much higher CP concentrations were necessary for a positive response in the SCG test than in the SCE test. PER was tested in the absence and in the presence of S9 mix and neither induced DNA migration nor increased SCE frequencies. In these experiments, a clear cytotoxic effect of PER was observed. To investigate a possible influence of DNA repair on the effects in the SCG test, cells were treated for 2 h and further incubated for 1 h after removal of the test substance. This procedure caused a clear decrease in induced DNA migration in experiments with Bap and CP, whereas no reduction was found with MNNG. This modified protocol did not lead to the detection of DNA effects after treatment with PER. The results indicate that the SCG test responds to various DNA lesions and does not seem to be sensitive to non-genotoxic cell killing. Its sensitivity obviously depends on the type(s) of induced DNA lesions and the effects can be modified by DNA repair processes in a complex manner. For the detection of genotoxic properties of chemicals with the in vitro SCG test, a single evaluation at the end of the exposure period seems to be sufficient.