Effect of methylxanthines on motility and fertility of frozen-thawed goat semen

We studied the effects of 2 methylxanthines (caffeine and theophylline) at different concentrations on goat sperm motility and live spermatozoa and on the percentage of acrosomal damage and fertility. Altogether, 144 semen samples collected from 12 bucks (3 each from Black Bengal and Beetal, and 6 from cross-breds) were diluted in TRIS extender, divided into 5 equal fractions; then caffeine and theophylline were added at 2 concentrations (2 and 5 mM) in different fractions. These samples were frozen in liquid nitrogen vapor, thawed at 37 degrees C for 15 sec, and evaluated for motility and other semen attributes. Addition of caffeine and theophylline had a stimulatory effect on goat spermatozoa. It was further observed that the effect of these agents was concentration-dependent, with 2 mM caffeine and 5 mM theophylline yielding the best results in respect to the percentage of motility in all 3 breeds of goats tested. Among the two methylxanthines used, caffeine was found to be the more effective in Improving motility than theophylline. There was no significant effect on the percentages of live spermatozoa and acrosomal damage due to the addition of these 2 methylxanthines to the extender. Fertility rates with Tris + 2 mM caffeine (60.20 %) and with Tris + 5 mM theophylline (58.88 %) extended semen were apparently higher than those with the Tris-diluted semen (50.0 %), although these differences were not significant.     

Theacrine (1,3,7,9-tetramethyluric acid) synthesis in leaves of a Chinese tea,kucha (Camellia assamica var. kucha)

Theacrine (1,3,7,9-tetramethyluric acid) and caffeine were the major purine alkaloids in the leaves of an unusual Chinese tea known as kucha (Camellia assamica var. kucha). Endogenous levels of theacrine and caffeine in expanding buds and young leaves were ca. 2.8 and 0.6-2.7% of the dry wt, respectively, but the concentrations were lower in the mature leaves. Radioactivity from S-adenosyl-L-[methyl-14C]methionine was incorporated into theacrine as well as theobromine and caffeine by leaf disks of kucha, indicating that S-adenosyl-L-methionine acts as the methyl donor not only for caffeine biosynthesis but also for theacrine production. [8-14C]Caffeine was converted to theacrine by kucha leaves with highest incorporation occurring in expanding buds. When [8-14C]adenosine, the most effective purine precursor for caffeine biosynthesis in tea (Camellia sinensis), was incubated with young kucha leaves for 24 h, up to 1% of total radioactivity was recovered in theacrine. However, pulse-chase experiments with [8-14C]adenosine demonstrated much more extensive incorporation of label into caffeine than theacrine, possibly because of dilution of [14C]caffeine produced by the large endogenous caffeine pool. These results indicate that in kucha leaves theacrine is synthesized from caffeine in what is probably a three-step pathway with 1,3,7-methyluric acid acting an intermediate. This is a first demonstration that theacrine is synthesized from adenosine via caffeine.     

Studies on DNA binding of caffeine and derivatives: evidence of intercalation by DNA-unwinding experiments

Caffeine and derivatives are compounds with pleiotropic effects on the genetic material which are supposed to originate from drugs binding to DNA. Here we show, by using two different topological methods, that methylated oxypurines, at biologically relevant concentrations, unwind DNA in a fashion similar to known intercalators. Methylated oxypurines could be ranked by decreasing unwinding potency: 8-methoxycaffeine greater than 8-ethoxycaffeine greater than 8-chlorocaffeine greater than caffeine greater than theophylline. These findings confirm, with a different assay, interaction of caffeine with DNA and add additional support to an intercalative mode of binding of these drugs to DNA.     

Changes induced by caffeine, theophylline and theobromine on calcium uptake, respiration and ATP levels in rat-liver mitochondria

1. The inhibition of calcium uptake and cellular respiration depend on the concentration of the compounds as shown by the concentration-effect curves. 2. The concentrations at which 50% inhibition of the transport of calcium takes place (caffeine 45 mM, theophylline 12 mM, theobromine, 4 mM) do not coincide exactly with those that produce the same effect on cellular respiration (caffeine 60 mM, theophylline 22 mM, theobromine 8 mM). 3. ATP concentrations under different conditions were also determined; a decrease in their value induced by the drugs was observed. No significant differences were observed, however, between the effect produced by the methyl-xanthines. 4. These findings suggest that these compounds are able to affect in some way the maintenance of energy gradients linked to the effects studied.     

Effects of G2 treatments with inhibitors of DNA synthesis and repair on chromosome damage induced by X-rays and chemical clastogens in root tips of Vicia faba. Comparison with corresponding effects in cultured human lymphocytes

The frequencies of chromatid aberrations produced in roots of Vicia faba by clastogenic (chromosome-damaging) agents were strongly enhanced by exposing the root-tip cells to inhibitors of DNA synthesis during the G2 phase. Chromosome damage produced by both S-dependent (maleic hydrazide, methyl methanesulfonate, thio-TEPA) and S-independent (X-rays, streptonigrin) mechanisms was enhanced by the inhibitor treatments. The types of aberrations affected by the inhibitors were mainly chromatid gaps and breaks and isochromatid breaks of the non-union type. Most effective among the inhibitors tested were hydroxyurea (HU) and 5-fluorodeoxyuridine (FdUrd). Post-treatments with caffeine were effective in enhancing clastogen-induced chromosome damage when given during the S phase. All types of aberrations, exchanges as well as breaks, were enhanced by the post-treatments. When given during the G2 phase, caffeine enhanced only the frequency of chromatid aberrations produced by X-rays. The enhancement was slight and obtained only when the cells were irradiated in the G2 phase and immediately post-treated with caffeine. Clastogen-treated cultures of human lymphocytes responded to post-treatments with inhibitors of DNA synthesis in very much the same way as clastogen-treated root-tip cells of Vicia faba. Thus, the frequencies of chromatid gaps and breaks and isochromatid breaks of the non-union type were strongly enhanced by exposing clastogen-treated lymphocytes to inhibitors of DNA synthesis during the G2 phase. The efficiency of the inhibitors, however, varied considerably in the two materials. On the whole, the number of inhibitors capable of enhancing induced chromosome damage was much larger in lymphocytes than in bean root tips. Only HU was equally effective in both materials. The most striking difference between the two materials was found when caffeine was given as a post-treatment. Thus, in human lymphocytes the frequencies of chromatid aberrations induced by most clastogenic agents were strongly enhanced when caffeine was given during the G2 phase, but little affected by post-treatments with caffeine during the S phase.     

Interaction of caffeine and of theophylline with liver glutamate dehydrogenase

Caffeine and theophylline inhibited the activity of rat liver glutamate dehydrogenase (GDH), but not that of beef liver GDH, in forward and reverse directions of the enzyme reaction. In the forward direction, approximately 16 mM caffeine or 16 mM theophylline inhibited 50 per cent of the rat liver GDH activity (I50); while in the reverse direction, the I50 of caffeine and theophylline was 15 mM and 8 mM, respectively. The inhibition produced by caffeine was cooperative in both directions, while that of theophylline was negatively cooperative in the forward direction and non-cooperative in the reverse. However, ADP reduced the inhibitory effect of caffeine and theophylline to the extent of 40% and 80%, respectively. The Ki values obtained for caffeine and theophylline were different in the presence of various concentrations of substrates and coenzymes. Based upon these data, we presume that certain subtle changes occurring in the conformation of the rat liver GDH (probably at the ADP/NADH site) in comparison with those of the beef liver GDH may be responsible for its inhibition by caffeine and theophylline.     

Metabolic Relations between Methylxanthines and Methyluric Acids in Coffea L

Metabolism of purine alkaloids in the leaves of Coffea dewevrei De Wild et Durand var excelsa Chev, Coffea liberica Bull ex Hiern and Coffea abeokutae Cramer was studied by analyzing leaf discs collected during vegetative development and by feeding the following radioactive tracers: [¹⁴C]theobromine, [¹⁴C]caffeine, and [¹⁴C]theacrine (1,3,7,9-tetramethyluric acid). Their principal metabolites were quantitatively and qualitatively determined. All three species convert the precursors to the same radioactive products, and proceed through the same four maturity stages characterized by the alkaloid accumulation pattern and by a particular transformation potency: (stage 1) young plant accumulating caffeine, transforms theobromine to caffeine; (stage 2) caffeine is gradually replaced by theacrine, theobromine and caffeine are converted to theacrine; (stage 3) theacrine disappears whereas liberine (O(2), 1,9-thrimethyluric acid) accumulates, theacrine is metabolized to liberine; (stage 4) branched-out plant containing liberine but no theacrine, caffeine is converted rapidly to liberine via theacrine. Methylliberine (O(2),1,7,9-tetramethyluric acid), presumably the direct precursor of liberine, is occasionally found in low concentrations at stage 3 and 4.

The collective term `liberio-excelsoid' introduced by geneticists for the numerous races or species of Pachycoffea is in accordance with the phytochemical equality found in this work.

     

In vitro response of goldfish (Carassius auratus L.) dermal melanophores to cyclic 3',5'-nucleotides, nucleoside 5'-phosphates and methylxanthines

cAMP, dbcAMP, cCMP, cGMP, theophylline and caffeine caused reversible melanosome dispersion within 5 minutes at 10 mM in the dermal melanophores of the black goldfish, Carassius auratus L. cTMP, cUMP, 5′-AMP, 5′-CMP, 5′-GMP, 5′-TMP, and 5′-UMP did not produce melanosome dispersion or aggregation in this melanophore system. cAMP was the most effective nucleotide in the induction of melanosome dispersion; at 10 mM, cGMP and at 5 mM, dbcAMP were the least effective of those nucleotides inducing melanosome dispersion. At the 10 mM level dbcAMP required 30 minutes to evoke the same degree of melanosome dispersion as 5 minutes cAMP treatment. Theophylline was more effective than caffeine in eliciting melanosome dispersion. At 1 mM, theophylline and caffeine first induced melanosome dispersion which was followed by aggregation in the course of the 30 minute test period. These reactions suggest both a high melanophore phosphodiesterase activity and competitive inhibition of phosphodiesterase by theophylline and caffeine. Induction of melanosome dispersion by several cyclic 3′,5′-nucleotides suggest multi-nucleotide control of melanosome dispersion. These findings also support a proposed mechanism of prostaglandin induced melanosome dispersion as well as the “second messenger” hypothesis.     

The effect of methylated oxypurines on the size of newly-synthesized DNA and on the production of chromosome aberrations after UV irradiation in Chinese hamster cells.

A comparison has been made, in Chinese hamster cells, of the ability of various methylated oxypurines to inhibit post-replication repair of DNA after UV irradiation and to potentiate UV-induced chromosome aberrations. DNA synthesized in UV-irradiated cells contains gaps, which are subsequently sealed by a process termed post-replication repair. In rodent cells this process is inhibited by caffeine and its analogues. This has been quantitated by measuring the molecular weight of the DNA synthesized in UV-irradiated cells during a 4-h pulse-labelling period in the presence or absence of inhibitors--the lower molecular weight the greater the inhibition. Eight methylated oxypurines were tested; caffeine and chlorocaffeine were always the most potent inhibitors, tetramethyluric acid was inactive, and the other five derivatives (methoxycaffeine, ethoxycaffeine, paraxanthine, theobromine and theophylline) had intermediate effects. Measurements of the potentiation of UV-induced chromosome aberrations showed that treatments with caffeine or chlorocaffeine again had the greatest effects, tetramethyluric acid and also theophylline had no potentiating activity, and methoxycaffeine was intermediate. This correlation between effects at the molecular and cytological levels is consistent with the hypothesis that the inhibition of post-replication repair by methylated oxypurines gives rise to the increased production of chromosome aberrations.     

Effect of theophylline and theobromine on cell respiration and calcium transport in isolated rat liver mitochondria

The influence of theophylline and theobromine on cellular respiration and on membrane transport of calcium has been studied in isolated rat liver mitochondria, using oxygen and Ca2+ selective electrodes. A linear decrease in respiratory coefficients, in the total amount and rate of "extra" oxygen consumption induced by ADP is observed with drug concentration. Theobromine does not show any appreciable effect on these respiratory parameters, but this result is similar to that observed with theophylline for the same concentration range. Calcium uptake coupled to respiration is inhibited by both drugs depending on their concentrations. Theobromine is more effective than theophylline. Calcium saturation of the mitochondria takes place in all cases after 36 +/- 2 s but only a 20% of the maximum calcium uptake observed in the absence of the drugs is determined in the presence of 15 mM theophylline or only 1.8 mM theobromine. Comparative studies show direct correlation between the pharmacological activities as stimulants of caffeine, theophylline and theobromine and their behaviour as inhibitors of calcium uptake coupled to respiration by mitochondria.     

Induction of Systemic Resistance in Pea to Pea Powdery Mildew by Exogenous Application of Salicylic Acid

Exogenous application of salicylic acid (SA) solutions to pea leaves induced systemic resistance to Erysiphe pisi. reducing by 20–30% the percentages of fungal germlings that successfully infected untreated leaves of SA-treated plants. SA concentrations of 1.5 and 15 mM were similarly effective, but 0.15 mM had no detectable effect. While 15 mM SA solutions were phytotoxic. 1.5 mM solutions caused no apparent damage indicating that resistance induction was not due to tissue damage. The induced resistance persisted for at least 13 days after treatment, but excision of treated leaves 1 day after SA application prevented full induction of systemic resistance, and the resistance was not expressed if untreated leaves were inoculated fewer than 3 days after SA application. The effect of SA was transmitted to leaves at nodes both above and below treated leaves. Chemical induction of systemic resistance may provide an additional means for controlling pea diseases.     

The inhibition by xanthine phosphodiesterase inhibitors of the induction of alkaline phosphatase activity in HeLa cells: relationship of enzyme activity to cyclic AMP concentrations.

The three xanthine derivatives, caffeine, theophylline and 3-isobutyl-1-methyl-xanthine (IBMX) produced dose-dependent increases in cyclic AMP concentrations in HeLa cells after long term treatment. Only IBMX produced increases over the first 60 minutes, with a peak of approximately 5-fold control values five to 10 minutes after the addition of the drug. About four hours after the addition of either 0.67 or 1.0 mM IBMX there was a second peak in the concentration of cyclic AMP which was at least as large and usually larger than the peak observed at five to ten minutes. Neither caffeine nor theophylline increased cyclic AMP concentrations above control values until one hour after addition of the compounds, and there was no indication of a peak in the concentration at four hours. Between 24 and 72 hours, all three compounds produced elevations in cyclic AMP levels that were steadily maintained. At any given concentration, the order of potency was IBMX greater than theophylline greater than caffeine. If the xanthine derivatives were removed from the medium after 24 hours of treatment, the cyclic AMP concentrations fell to control levels within one hour. Treatment with 5-iodo-2'-deoxyuridine (IdUrd) or hydrocortisone alone did not change the levels of cyclic AMP, nor did the presence of these inducers of alkaline phosphatase activity alter the effects of the xanthine derivations on cyclic AMP concentrations. The data showed a significant correlation between the magnitude of the increase in cycli AMP concentrations over the period from 24 to 72 hours and the degree of inhibition by the xanthine derivatives of the induction of alkaline phosphatase activity.     

The effect of glucose on liver glycogen synthase phosphatase activity in the presence of ATP-Mg

Glycogen particle synthase phosphatase activity is stimulated by glucose with an A0.5 of approximately 27 mM. The A0.5 is higher than the usual concentrations present in the liver. However, in vitro, certain methylxanthines such as caffeine or theophylline reduce the glucose A0.5 to approximately 10 mM, a concentration well within the normal range of liver glucose concentrations. Methylxanthines do not affect the maximum stimulation by glucose (2.3-fold greater than control rate). The phosphatase reaction also is inhibited by ATP-Mg (I0.5 = 0.1 mM). In the present studies, we have determined the interaction of these effectors. The presence of ATP-Mg at a concentration of 3 mM only slightly reduced the maximal stimulation by glucose. The A0.5 for glucose was unaffected (24 mM). The synergistic effect of caffeine with glucose also was not changed by the presence of ATP-Mg. The A0.5 for glucose was reduced to 11 mM, similar to that in the absence of ATP-Mg. In addition, maximum stimulation by glucose was unchanged. Similar results were obtained when theophylline replaced caffeine. We conclude that the ATP-Mg binding site on either the phosphatase or its substrate, synthase D, does not influence the glucose and methylxanthine binding sites. Effectively, ATP-Mg increased the range over which glucose stimulates the phosphatase activity. In the presence of ATP-Mg, the maximum stimulation by glucose is approximately 7-fold; whereas, in the absence of ATP-Mg it is approximately 2.3-fold. Thus, ATP-Mg may serve to increase the sensitivity of the synthase phosphatase reaction to glucose regulation under in vivo conditions.     

The influence of analogs of xanthine on the mutagenic effect of ultraviolet light

Summary Eight compounds can be added to those purine analogs which give synergistic effects with ultraviolet light: 1,3,7,9-tetra-methylxanthine methyl sulfate, 8-chlorocaffeine, ethyltheophylline, 6-dimethylaminopurine, 6-methoxypurine, 8-ethoxycaffeine, 1,3-dimethyl uric acid and 3-methyl uric acid. It appears likely that these are merely representatives of the class of methyl purines, and that other compounds are also active. The synergistic activity varies among the compounds investigated; some give a synergistic effect slightly higher than caffeine, but apparently act by the same mechanism. The height of effect of all of the synergistic purine analogs has been correlated with the stability of the structures determined by inductive effects and steric hindrance. The lack of activity with pyrimidines (l-methyl thymine and 1,3-dimethyluracil) leads to the conclusion that both rings (7,8,9 positions) of the purine molecule are necessary for the synergistic effect.This work was supported by the National Institutes of Health Research Grant AI-05340, the University of Kansas General Research Fund, and an N.I.H. Training Grant (5 T1 GM-703) to the Department of Microbiology.     

Inhibitors in tea of intestinal absorption of phenylalanine in rats

This work studies the effect of tea extract on the mucosal and serosal transport of phenylalanine, and attempts to identify the active ingredient(s) therein by studying the effect of known tea constituents like theophylline, caffeine and tannic acid. Tea and all the constituents tested inhibited the mucosal uptake of phenylalanine. The serosal transport was unaffected by caffeine and tannic acid, but inhibited by theophylline and high concentrations of tea. The in vitro activity of the intestinal Na⁺-K⁺ ATPase was also assayed from a jejunal homogenate in presence of theophylline, caffeine, tannic acid and cAMP. All were found to inhibit significantly the enzyme. The in vitro activity of a purified Na⁺-K⁺ ATPase was however stimulated by theophylline and caffeine, and inhibited only by tannic acid. It was concluded that the inhibitory effect of tea is exerted mainly through its constituents which inhibit the Na⁺-K⁺ pump directly (tannic acid) or indirectly (theophylline and caffeine), possibly by elevating cAMP levels, dissipating thus the sodium gradient needed for the mucosal uptake of the amino acid.     

Mutagenicity of methylisocyanate and its reaction products to cultured mammalian cells

Methylisocyanate (MIC) induced mutagenic responses in the absence of exogenous activation in the mouse lymphoma cell forward mutation assay at concentrations as low as 8-24 microM. MIC produced predominantly small mutant colonies, suggesting the possibility of clastogenic activity. The intermediate hydrolysis product, methylamine, was also mutagenic without exogenous activation but required several hundred-fold higher concentrations (ca. 3 mM). N,N'-Dimethylurea, the final product in the reaction of methylisocyanate and water, was totally refractory in either the presence or absence of S9 for concentrations up to 57 mM (5 mg/ml). The ethyl ester of N-methylcarbamic acid was also tested since it was the only available analogue to the highly reactive N-methylcarbamic acid intermediate. This compound was mutagenic only in the presence of S9 at doses exceeding 5-40 microM, which suggested the possibility that the free acid, produced by enzymatic hydrolysis, is also mutagenic. The mutagenic activity of the ester resulted solely in the production of small mutant colonies.     

Involvement of cAMP in initiating maturation of the brittle-star Amphipholis kochii oocytes: induction of oocyte maturation by inhibitors of cyclic nucleotide phosphodiesterase and activators of adenylate cyclase

The maturation of brittle-star (Amphipholis kochii) oocytes, i.e., the reinitiation of meiosis accompanied by germinal vesicle breakdown (GVBD) and the acquisition of fertilizability, was induced by acid (pH 3.0) seawater containing 10 mM cAMP. Oocyte maturation was also induced by seawater of normal pH (pH 8.0) that contained either an inhibitor of cyclic nucleotide phosphodiesterase (25 mM theophylline, 25 mM caffeine) or an activator of adenylate cyclase (100 microM forskolin, 0.6 microM cholera toxin). Experiments in which the oocytes were treated with forskolin or theophylline for various periods of time demonstrated that there was a positive correlation between the oocyte cAMP level measured by radioimmunoassay and the extent of GVBD induced in each treatment: both increased as the treatment period became longer and about a threefold increase in cAMP level induced 50% GVBD. These results indicate that an increase in cAMP level initiates maturation of the brittle-star oocytes.     

Relative efficacies of indole antioxidants in reducing autoxidation and iron-induced lipid peroxidation in hamster testes

Increased iron stores are associated with free radical generation and carcinogenesis. Lipid peroxidation is involved in DNA damage, thus indirectly participating in the early steps of tumor initiation. Melatonin and structurally related indoles are effective in protecting against oxidative stress. The aim of the study was to compare the relative efficacies of melatonin, N-acetylserotonin (NAS), indole-3-propionic acid (IPA), and 5-hydroxy-indole-3-acetic acid (5HIAA) in altering basal and iron-induced lipid peroxidation in homogenates of hamster testes. To determine the effect of the indoles on the autoxidation of lipids, homogenates were incubated in the presence of each agent in concentrations of 0.0, 0.01, 0.05, 0.1, 0.25, 0.5, 0.75, 1.0, 2.0, 2.5, or 5.0 mM. To study their effects on induced lipid peroxidation, homogenates were incubated with FeSO(4) (30 microM + H(2)O(2) (0.1 mM) + each of the indoles in the same concentrations as above. The degree of lipid peroxidation was expressed as concentrations of malondialdehyde + 4-hydroxyalkenals (MDA + 4-HDA) per mg protein. The indoles decreased both basal and iron-related lipid peroxidation in a concentration-dependent manner. Melatonin reduced basal MDA + 4-HDA levels when used at the concentrations of 0.25 mM or higher, and prevented iron-induced lipid peroxidation at concentrations of 1.0, 2.0, 2.5, or 5.0 mM. The lowest effective concentrations of NAS required to lower basal and iron-related lipid peroxidation were 0.05 mM and 0.25 mM, respectively. IPA, only when used in the highest concentrations of 2.5 mM or 5 mM inhibited basal lipid peroxidation levels and it was ineffective on the levels of MDA + 4-HDA due to iron damage. 5HIAA reduced basal lipid peroxidation when used at concentrations of 0.25 mM or higher, and it prevented iron-induced lipid peroxidation only at the highest applied concentration (5 mM). In conclusion, melatonin and related indoles at pharmacological concentrations protect against both the autoxidation of lipids as well as induced peroxidation of lipids in testes. In doing so, these agents would be expected to reduce testicular cancer that is initiated by products of lipid peroxidation.     

Clastogenic effects of dietary supplement--Spirulina alga, and some medicinal plant products from Boswellia serrata, Withania somnifera on mice

Pretreatment of aqueous extracts of Zyrulina (Spirulina), Aswagandha (Withania) and Nopane (Boswellia) on colchicine induced chromosome damage showed weakness of clastogenic activity in Swiss albino mice. None of the treatments increased significantly the number of chromosome aberrations.     

Chromosomal aberrations and sister-chromatid exchanges induced by N-nitroso-2-acetylaminofluorene and their modifications by arsenite and selenite in Chinese hamster ovary cells.

The frequencies of chromosomal aberrations (CA) and sister-chromatid exchanges (SCE) in Chinese hamster cells were significantly increased by the direct-acting mutagen N-nitroso-2-acetylaminofluorene (N-NO-AAF) at the concentration of 0.1 mM. N-NO-AAF was prepared by nitrosation of the protohepatocarcinogen 2-acetylaminofluorene. The induced CA, which included chromatid breaks, chromatid exchanges, chromosome breaks, and chromosome ring formation were significantly potentiated by the presence of sodium arsenite (10 microM), but not by hydroxyurea (20 mM) or cytosine arabinoside (25 microM). On the other hand, the clastogenic effect of N-NO-AAF was effectively inhibited by sodium selenite (100 microM). Arsenite (10 microM) was shown to be moderately active in CA induction which was partially blocked by the presence of selenite (10 nM). N-Nitroso compounds such as N-nitroso-N-methylurea, N-nitroso-N-ethylurea and N-methyl-N'-nitro-N-nitrosoguanidine were equally or more active in the induction of CA and SCE in CHO cells when compared with N-NO-AAF. The cell cycle was significantly delayed by the intervention of N-NO-AAF.