Effect of 5-fluoro-2'-deoxyuridine (FdUrd) on 5-bromo-2'-deoxyuridine (BrdUrd) incorporation into DNA measured with a monoclonal BrdUrd antibody and by the BrdUrd/Hoechst quenching effect

The purpose of this study was to improve the application of bromodeoxyuridine (BrdUrd) for the flow cytometric analysis of cell kinetics. In order to obtain a quantitative measure of the DNA synthesis rate (or the number of divided cells), BrdUrd should replace thymidine (dThd) completely in the newly synthesized DNA strands. The de novo synthesis of dThd monophosphate competing with BrdUrd incorporation was stopped by fluorodeoxyuridine (FdUrd). Cells of a human leukemic cell line (REH) were exposed to BrdUrd for either 20 min, 8 h, or 24 h. Bromodeoxyuridine incorporation was determined by a monoclonal antibody as well as by the BrdUrd/Hoechst (H) technique. Counterstaining of the DNA was performed with propidium iodide or ethidium bromide. DNA fluorescence was measured in both techniques with a two-parameter flow cytometer, the histograms being analyzed by computer. It was found that FdUrd is required in the BrdUrd/H technique for replacement of dThd at low BrdUrd concentrations and long incubation times. With short incubation periods, as used for detection by the monoclonal anti-BrdUrd antibody, FdUrd increases the incorporated BrdUrd amount when BrdUrd concentrations of 10 microM or less are applied.     

The effect of single versus double-strand substitution on halogenated pyrimidine-induced radiosensitization and DNA strand breakage in human tumor cells

To better understand the mechanism underlying halogenated pyrimidine-mediated cytotoxicity and radiosensitization in human tumor cells, a study was undertaken to determine the influence of unifilar (one DNA strand) versus bifilar (both DNA strands) substitution of thymidine by the halogenated bases 5-iodo-2'-deoxyuridine (IdUrd) and 5-bromo-2'-deoxyuridine (BrdUrd) in HT29 human colon cancer cells. Unifilar labeling was obtained by incubating cells with IdUrd or BrdUrd for one doubling time. Cells were incubated for at least three doublings to approximate bifilar substitution. Only IdUrd caused significant cytotoxicity, which correlated with incorporation into DNA. Both BrdUrd and IdUrd were potent radiosensitizers. Radiosensitization was linearly correlated with incorporation of both bases regardless of the number of strands in which thymidine was substituted. In contrast, the relationship between radiosensitization and DNA double-strand breakage was critically dependent in the case of IdUrd, but not for BrdUrd, on whether substitution was unifilar or bifilar. These findings suggest that incorporation is the best predictor of radiation sensitivity, and that the induction of DNA double-strand breaks alone does not account for radiosensitization mediated by halogenated pyrimidines in these human tumor cells.     

Breakage of Parental DNA Strands in Haemophilus influenzae by 313 nm Radiation after Replication in the Presence of 5-Bromodeoxyuridine

Haemophilus influenzae was labeled with thymidine-³H (dThd), then grown in the presence of 5-bromodeoxyuridine (BrdUrd), and then irradiated with 313 nm light (a wavelength that selectively photolyzes DNA containing 5-bromouracil [BrUra]). Irradiation with 313 nm light induced breaks in the ³H-labeled strands in cells grown with BrdUrd at a much higher frequency than in ¹⁴C-labeled DNA of cells not exposed to BrdUrd. Breakage of the ³H-labeled strands was about 0.6% as efficient as that of fully BrUra-substituted DNA. During growth in the presence of BrdUrd, susceptibility to 313 nm-induced breakage of the ³H-labeled DNA strands increased, reaching a maximum in about one generation, and it decreased to zero during subsequent growth for one generation in medium containing dThd instead of BrdUrd. Heat denaturation of DNA extracted from dThd-³H-labeled cells grown in the presence of BrdUrd eliminated 313 nm-induced breakage of the ³H-labeled strands. It is concluded that breakage of the ³H-labeled DNA strands resulted from reaction with photoproducts in the base-paired, BrUra-containing strands, rather than from photolysis of BrdUrd incorporated into parental strands. It may be possible to utilize the phenomenon of interstrand breakage in physical studies of DNA replication.     

BrdUrd incorporation studies for evaluation of spermatogenesis in the blue fox.

The utility of BrdUrd incorporation techniques for studies of spermatogenesis was investigated in the blue fox. BrdUrd was injected intraperitoneally followed by collection of testicular tissue by castration/hemicastration at intervals up to 35 days after pulse labelling. Fluorescent tagged monoclonal antibodies against BrdUrd allowed detection of cells with incorporated tracer in histological sections by fluorescent light microscopy as well as in isolated testicular cells by bivariate BrdUrd/DNA flow cytometry. The duration of the spermatogenic cycle was estimated by following the labelled cohort of preleptotene spermatocytes by immunofluorescence in sections through the various stages of maturation to the late spermatid stage. These data were confirmed by bivariate BrdUrd/DNA flow cytometry of testicular cells in suspensions. Furthermore, estimations of the S phase durations and length of the spermatogonial cell cycle were possible. A consistent and satisfactory fluorescence intensity of incorporated label throughout the study shows that degradation of the incorporated label is no practical problem for this type of study, and suggests that the method is an excellent tool for studying aspects of proliferation and maturation during normal as well as perturbed spermatogenesis. Advantages of the described method include avoidance of potential radiation influence on spermatogenesis from commonly used radiolabelled tracers, e.g., 3H-TdR, and that both large and small animals can be investigated at modest cost since the unlabelled BrdUrd is considerably less expensive than labelled tracers.     

Production of DNA bifilarly substituted with bromodeoxyuridine in the first round of synthesis: branch migration during isolation of cellular DNA.

Incubation of human lymphoid cells with bromodeoxyuridine (BrdUrd) for short periods produces three classes of DNA containing analog: DNAHL (hybrid DNA, density approximately equal to 1.75 g/cm3), DNAint (intermediate density DNA, density approximately equal to 1.71 g/cm3), and DNAHH (DNA with both strands containing analog, density approximately equal to 1.80 g/cm3). Preparations of DNAint yield DNAHH after extensive shearing and/or treatment with single strand specific endonuclease. Cross-linking of pulse-labeled (BrdUrd + 3HdT) DNA in cells by treatment with trioxsalen and near UV light before lysis prevents the appearance of DNAHH.Cross-linking after lysis has little effect. A large fraction of DNAHH is obtained after incubation of cells with caffeine. Extraction of DNA at high salt concentration or cross-linking with trioxsalen and near UV light drastically reduced the amount of DNAHH obtained from caffeine-treated cells. We conclude that most DNAHH arises from in vitro branch migration in isolated DNA growing points.     

Measurement of SCE frequencies in plants: a simple Feulgen-staining procedure for vicia faba

A relatively straightforward approach is described to obtain differential contrast in sister chromatids for SCE detection in Vicia faba after BrdUrd incorporation. The hydrolysis time of the well-known Feulgen reaction was extended to differentially degrade the DNA, the BrdUrd-substituted strands being more resistant. The procedure may easily be adapted for other plant species with large chromosomes.     

Formation of Okazaki fragments in polyoma DNA synthesis caused by misincorporation of uracil.

When dUTP replaced dTTP during polyoma DNA replication in isolated cell nuclei, radioactivity from labeled deoxynucleoside triphosphates was almost exclusively recovered in very short Okazaki fragments and incorporation ceased after a short time. Addition of uracil, a known inhibitor of the enzyme uracil-DNA glycosidase (Lindahl et al., 1977), increased total synthesis and shifted the incorporation to longer progeny strands. The presence of as little as 2.5% of dUTP in a dTTP-containing system gave a distinct increase in isotope incorporation into Okazaki pieces accompanied by a corresponding decrease in longer strands. This effect was reversed completely by uracil. The short strands formed from dUTP could be chased efficiently into long strands. Our results suggest that dUTP can be incorporated in place of dTTP into polyoma DNA, and that polyoma-infected nuclei, similar to E. coli (Tye et al., 1977), contain an excision-repair system which by removal of uracil causes strand breakage and under certain circumstances may contribute to the formation of Okazaki fragments.     

Effect of Caffeine on DNA Synthesis in Mammalian Cells

Alkaline sucrose sedimentation studies of DNA from mouse lymphoma cells (L5178Y) treated with caffeine have demonstrated the following effects. Caffeine (at a concentration of 1.6 mM) does not introduce strand breaks into preformed DNA nor does it inhibit the rejoining of γ-ray-induced strand breaks. Although it does not affect the over-all rate of DNA synthesis, pulse labeling experiments show that the DNA strands synthesized in its presence are smaller than those made in its absence. This could be the result of (a) DNA being made in shorter replicating units or (b) small gaps in the daughter DNA strands within normal-sized replicating units. These two alternative models were indirectly distinguished as follows. After a pulse label with thymidine-³H in the presence of caffeine, cells were incubated without caffeine in bromodeoxyuridine (BrdUrd). During this incubation, growing strands are elongated and hypothetical gaps (model b) filled in with bromuracil (BrUra)-substituted DNA. The BrUra-containing DNA segments will now be of different lengths on the two models. With smaller replicating units (a) the “elongation segments” will be somewhat smaller than but the same order of magnitude as those in untreated cells, whereas with small gaps (b) the “filled-in gap segments” would be expected to be at least an order of magnitude smaller. The BrUra-containing regions of DNA can be selectively broken open by exposing the cells to light at 313 nm. The exposure required to break open the BUra-substituted regions is inversely related to, and hence gives a measure of, the size of these regions. In caffeine-treated cells these regions were found to be somewhat smaller than but of comparable size with those in untreated cells; this is consistent with the DNA being synthesized in smaller units and argues against the presence of small gaps in the daughter strands.     

Evidence for ethylation of rat liver deoxyribonucleic acid after administration of ethionine

1. Administration of a large dose (500mg/kg body wt.) of (3)H-labelled l-ethionine to rats resulted in the incorporation of a small amount of radioactivity into the liver DNA. Considerable evidence that this radioactivity was not due to contamination of the isolated DNA with labelled protein, RNA, S-adenosyl-l-ethionine or l-ethionine was obtained. 2. After acidic hydrolysis of the DNA isolated from the livers of rats treated with labelled l-ethionine, virtually all of the radioactivity present in the DNA was found in a fraction with similar chromatographic properties to 7-ethylguanine. 3. Treatment of rats with comparable doses of l-methionine did not lead to the formation of 7-methylguanine in the liver DNA. 4. These results are discussed in relation to the induction of liver tumours by ethionine.     

The incorporation of 5-bromodeoxyuridine into DNA of the area opaca vasculosa of chick embryos

The incorporation of 5-bromodeoxyuridine (5-BrdUrd) into DNA of the area opaca vasculosa (AOV) of chick embryos during organ culture was measured. The AOV from blastoderms of the definitive primitive streak stage will not form red cells in the presence of BrdUrd while the AOV of 1–3 somite blastoderms is unaffected by the presence of 5-BrdUrd. About 90% of the original non-density labeled DNA can replicate in the presence of 5-BrdUrd if the tissues come from the younger sensitive embryos, but only 65% of the original DNA will replicate from tissues of older insensitive embryos. Tissues from embryos of both ages replace about 80% of the thymidine by BrdUrd in each newly synthesized strand of DNA; tissues from embryos of both ages will form DNA of hybrid density after one cell generation, and will also form double-heavy DNA after longer periods of culture in the presence of 5-BrdUrd. During recovery from 5-BrdUrd inhibition during a thymidine chase, the density-labeled DNA is replicated so that the new DNA of normal density is formed, but the original heavy 5-BrdUrd containing strands are conserved. It is suggested that inhibition of red cell formation by 5-BrdUrd may occur by incorporation of 5-BrdUrd into DNA of endoderm cells, rather than by acting only directly on red cell precursors.     

Characterization of the enhancing effect of caffeine on sister-chromatid exchanges induced by ultraviolet radiation in excision-proficient xeroderma pigmentosum lymphoblastoid cells

Cells of some excision-proficient xeroderma pigmentosum (XP) cell lines are highly sensitive to post-UV caffeine treatment in terms of sister-chromatid exchange (SCE) induction as well as cell lethality. In the present study, we conducted a detailed investigation of the enhancing effect of caffeine on SCE frequency induced by UV in excision-proficient XP cells, and obtained the following results. (1) Continuous post-UV treatment with 1 mM caffeine markedly enhances UV-induced SCEs and such enhanced SCEs occur with similar frequency during either the 1st or the 2nd cell cycle in the presence of caffeine and 5-bromodeoxyuridine (BrdUrd). (2) The high sensitivity of the cells to post-UV caffeine treatment persists for at least 2 days after UV when irradiated cells are held in either the proliferating or the nonproliferating state prior to the addition of BrdUrd. (3) Caffeine exerts its effect on cells in S phase. (4) Neither BrdUrd in the medium nor the incorporated 5-bromodeoxyuridine monophosphate (BrdUMP) in DNA plays an appreciable role in the expression of the enhancing effect of caffeine. The most likely explanation for our findings is as follows. In excision-proficient XP cells, the cause of SCE formation such as UV-induced lesions or resulting perturbations of DNA replication persists until the 2nd round or more of post-UV DNA replication. If caffeine is given as post-UV treatment, such abnormalities may be amplified, resulting in a synergistic increase in SCE frequency.     

Aphidicolin-Resistant Mutants of Mouse Lymphoma L5178y Cells with a High Incidence of Spontaneous Sister Chromatid Exchanges

Two aphidicolin-resistant cell mutants (AC 12 and AC 41) with a fourfold increase in spontaneous frequency of sister chromatid exchanges (SCEs) were obtained out of over 400 aphidicolin-resistant mutants isolated from mouse lymphoma L5178Y cells. They also exhibited three- to fourfold increases in spontaneous frequency of chromosome aberrations (CAs). To determine whether the high level of SCE frequency in AC 12 is caused by 5-bromodeoxyuridine (BrdUrd) used for visualizing SCEs, the effect of BrdUrd incorporated into DNA on SCE induction was analyzed. The SCE frequencies in AC 12 remained constant at BrdUrd incorporation levels corresponding to 2-90% substitution for thymidine in DNA. In addition, the small amount of BrdUrd incorporated into both daughter and parenteral DNA strands in AC 12 had minimal effect on SCE induction. Furthermore, AC 12 and AC 41 were slightly resistant to BrdUrd with respect to the induction of CAs, the inhibition of cell-cycle progression and the decrease in mitotic activity. These findings suggest that the high incidence of SCEs in AC 12 and AC 41 is formed by their intrinsic defects, not by the effects of BrdUrd used. The analysis of SCE frequencies in hybrid cells between these mutants and the parental L5178Y revealed that the genetic defects in AC 12 and AC 41 appear to be recessive, and that these two mutants belong to the same complementation group. Furthermore, AC 12 belonged to a different complementation group from ES 4, which was isolated previously from L5178Y as an SCE mutant with a twofold higher frequency of spontaneous SCEs. This finding indicates that at least two different genetic defects participate in the formation of the high incidence of spontaneous SCEs in mouse cells. These SCE mutants would provide valuable cell materials for studying the molecular mechanism of SCE formation.     

Enhancement of chromosome aberrations by the combination of DNA substitution with halogenated deoxyuridine and streptonigrin treatments

We treated CHO cells with streptonigrin (SN) alone, in combination with BrdUrd or IdUrd substitution, and with or without the addition of caffeine. The cells assessed for chromosome damage by SN were in the G₂ period and the magnitude of the damage was expressed as monosubstituted chromatid breaks, bisubstituted chromatid breaks and boundary regions breaks (boundary regions indicate the point of exchange of mono- and bisubstituted chromatids). We found that the combination of BrdUrd or IdUrd substitution with SN treatments produced a remarkable increase in the frequency of breaks over the frequencies observed with the halogenated compound only. The effect was more evident with IdUrd than with BrdUrd, and more dramatic in bisubstituted than in monosubstituted chromatids. The frequency of boundary breaks in cells treated with BrdUrd plus SN was similar to the frequency of breaks in monosubstituted chromatids treated similarly. Conversely, the damage in boundary regions was almost similar to that in bisubstituted chromatids in cells challenged with IdUrd plus SN. The addition of caffeine to BrdUrd-substituted chromosomes gave rise to a marked enhancement of breakages with a gradient of chromatid damage that was: bisubstituted > monosubstituted > boundary regions. A further increase of chromatin breaks maintaining the gradient indicated above was obtained when the cells were treated with BrdUrd plus SN plus caffeine. We propose that BrdUrd and IdUrd substitution alone or in combination with caffeine treatments and with SN in its capacity to bind DNA, give rise to different chromatin structures capable of modulating the DNA damage induced along the chromatin fibril by the active oxygen species liberated by SN-DNA complexes.     

Reduced N-methyl-N′-nitro-N-nitrosoguanidine sister chromatid exchange induction in chinese hamster V79 cells pre-exposed to 5-bromodeoxyuridine

The sequence in which N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and 5-bromodeoxyuridine (BrdU) are added to cell cultures affects the number of sister chromatid exchanges (SCE) induced by MNNG. When V79 Chinese hamster cell monolayer cultures were treated with MNNG for 2 h prior to addition of BrdUrd, approximately a 4–5-fold increase in SCE was observed at the second division metaphases compared to controls exposed to BrdU alone. This effect was independent of whether one or three DNA strands had been substituted as a result of incubating the cells through one or two DNA synthesis periods in the presence of BrdU. This increase in SCE also occurred after MNNG exposure and BrdU incubation was extended for three division cycles. In contrast, when BrdU incorporation preceded MNNG treatment, the average number of SCE/metaphase was reduced 70–80% at the second division cycle and 60% relative to the total number found in three division cycles. SCE induction by MNNG does not involve a caffeine sensitive step since caffeine had no effect on the SCE frequency regardless of the treatment protocol. The conditions in which BrdU preceded MNNG exposure may be responsible for either reducing the number of DNA sites available for interaction with MNNG or preventing the expression of SCE.     

Bromodeoxyuridine/DNA analysis of replication in CHO cells after exposure to UV light

The effects of ultraviolet light on cellular DNA replication were evaluated in an asynchronous Chinese hamster ovary cell population. BrdUrd incorporation was measured asa function of cell-cycle position, using an antibody against bromodeoxyuridine (BrdUrd) and dual parameter flow cytometric analysis. After exposure to UV light, there was an immediate reduction ( 50%) of BrdUrd incorporation in S phase cells, with most of the cells of the population being affected to a similar degree. At 5 h after UV, a population of cells with increased BrdUrd appeared as cells that were in G1 phase at the time of irradiation entered S phase with apparently increased rates of DNA synthesis. For 8 h after UV exposure, incorporation of BrdUrd by the original S phase cells remained constant, whereas a significant portion of original G1 cells possessed rates of BrdUrd incorporation surpassing even those of control cells. Maturation rates of DNA synthesized immediately before or after exposure by alkaline elution, were similar. Therefore, DNA synthesis measured in the short pulse by anti-BrdUrd fluorescence after exposure to UV light was representative of genomic replication. Anti-BrdUrd measurements after DNA damage provide quantitative and qualitative information of cellular rates of DNA synthesis especially in instances where perturbation of cell-cycle progression is a dominant feature of the damage. In this study, striking differences of subsequent DNA synthesis rates between cells in G1 or S phase at the time of exposure were revealed.     

Biosynthesis of Caffeine in Flower Buds of Camellia sinensis

The biosynthesis of purine alkaloids in flower buds of tea plantswas investigated. More than 25% of total radioactivity of [8-¹⁴C]adeninetaken up by stamens isolated from tea flower buds was foundto have been incorporated into purine alkaloids, namely, theobromineand caffeine, 24 h after administration of the labelled compound.Pulse-chase experiments indicated that [8-¹⁴C]adenine takenup by the stamens was converted to adenine nucleotides and subsequentlyincorporated into theobromine and caffeine. Since 5 µMcoformycin, an inhibitor of AMP deaminase, inhibited the incorporationof radioactivity into the purine alkaloids, synthesis of caffeinefrom adenine nucleotides seems to be initiated by the reactionof AMP deaminase. Although most of the radioactivity from [8-¹⁴C]inosinewas recovered as CO₂ and ureides, considerable amounts of radioactivitywere recovered as purine alkaloids. The incorporation of radioactivityfrom [8-¹⁴C]inosine into the purine alkaloids was not affectedby coformycin. The five enzymes involved in synthesis of 5-phosphoribosyl-1-pyrophosphatefrom glucose were present in the stamens and petals of tea flowerbuds. From present and previous results, the pathway for thebiosynthesis of caffeine from adenine nucleotides in flowerbuds of tea is discussed.Copyright 1993, 1999 Academic Press Camellia sinensis, tea, stamen, flower, biosynthesis, purine alkaloids, caffeine, theobromine, adenine nucleotides, nucleotide biosynthesis     

Replication of mammalian DNA in bromodeoxyuridine: Appearance of a component with intermediate density

Summary When three lines of mammalian cells were cultured with 5-bromodeoxyuridine (BrdUrd) for less than one generation, their DNAs displayed three peaks in CsCl gradients. In addition to the expected unsubstituted (LL) and hybrid (LH) peaks, there was a significant absorbance peak of intermediate density (INT) between LH and LL DNAs. This INT DNA has characteristics expected of an intermediate of DNA replication. Upon shearing, it behaves as though it contains contiguous segments of unsubstituted and hybrid DNAs. Upon continuous exposure of cells to [³H]-BrdUrd, radioactivity accumulates in INT DNA for 60–90 min when a steady state condition is reached. At that time, the rate of incorporation into LH DNA increases, consistent with a precursorproduct relationship. In a pulse-chase experiment, radioactivity is chased from INT DNA into LH DNA. To account for the above observations and for the size and sharpness of the INT DNA peak in CsCl, we suggest that a high molecular weight replication intermediate accumulates before completing replication into mature daughter molecules.     

Thymidine metabolism and DNA synthesis in Newcastle disease virus-infected cells.

The inhibition of thymidine incorporation into DNA in Newcastle disease virus-infected cells has been studied. At 6 h after infection of L-929 cells at high multiplicity, transport of exogenous thymidine across the cell membrane was inhibited. The kinetics of this inhibition, decreased Vmax with no change in Km, suggest that there are fewer sites available for transport in infected cells. The conversion of thymidine to dTTP was not inhibited. Equilibrium of exogenous thymidine with the acid-soluble pool occurred more slowly and at a lower level of radioactivity than in uninfected cells, and there was a reduction in the rate of incorporation of exogenous thymidine into DNA. The reduction of incorporation into the pool and into DNA was proportionate. The size of total cellular dTTP pools was changed very little in infected cells. DNA synthesized in infected cells in the presence of [3H]BrdUrd had reduced incorporation of tritium but similar buoyant density to that from uninfected cells. The results show that Newcastle disease virus inhibits DNA synthesis directly and, in addition, decreases thymidine transport. Together these account for the overall decrease in thymidine incorporation into DNA of infected cells.     

THE EFFECT OF METHIONINE SULPHOXIMINE ON THE INCORPORATION OF LABELLED GLUCOSE, ACETATE, PHENYLALANINE AND PROLINE INTO GLUTAMATE AND RELATED AMINO ACIDS IN THE BRAINS OF MICE

—The incorporation of radioactivity from labelled glucose, acetate, phenylalanine and proline into glutamate, aspartate and glutamine was measured in mice treated with methionine sulphoximine and in the control animals. The labelled precursors were injected and their incorporation determined before the onset of convulsions. The incorporation of radioactivity from labelled glucose into the dicarboxylic amino acids was reduced, in particular the incorporation into glutamine. The incorporation of radioactivity from labelled acetate and phenylalanine into glutamate and aspartate was increased by methionine sulphoximine, while the incorporation into glutamine was not changed very much. The labelling of glutamine, relative to glutamate, was reduced with all precursors, indicating that glutamine synthetase was inhibited in vivo by methionine sulphoximine. It is very likely that methionine sulphoximine affects many aspects of energy metabolism in brain; in particular the metabolism of glucose seems to be inhibited, while the rate of conversion of substrates other than glucose seems to be increased.     

A method to measure the duration of DNA synthesis and the potential doubling time from a single sample

A method is described whereby the DNA synthesis time, Ts, can be calculated using data of a single sample of cells taken several hours after labelling with bromodeoxyuridine (BrdUrd). The method involves a simple calculation using flow cytometry data of BrdUrd incorporation (green fluorescence, FITC-labelled anti-BrdUrd-DNA antibody) and total DNA content (red fluorescence, propidium iodide). The movement of BrdUrd-labelled cells through the S phase can be quantified by measuring their mean red fluorescence relative to that of G1 and G2 cells. Assuming the movement of the labelled cells toward G2 is linear with time, Ts can be calculated by measuring their relative movement at any one time. The method was tested on cells in vitro and on bone marrow and tumor cells in vivo. Reasonable agreement was seen with published estimates of Ts for these tissues.