Distribution and Pathogenicity of Colletotrichum Species Associated With Mango Anthracnose in Mexico.

Mango anthracnose, caused by Colletotrichum spp., is the most significant disease of mango (Mangifera indica L.) in almost all production areas around the world. In Mexico, mango anthracnose has only been attributed to C. asianum and C. gloeosporioides. The aims of this study were to identify the Colletotrichum species associated with mango anthracnose symptoms in Mexico by phylogenetic inference using the ApMat marker, to determine the distribution of these species, and to test their pathogenicity and virulence on mango fruits. Surveys were carried out from 2010 to 2012 in 59 commercial orchards in the major mango growing states of Mexico, and a total of 118 isolates were obtained from leaves, twigs, and fruits with typical anthracnose symptoms. All isolates were tentatively identified in the C. gloeosporioides species complex based on morphological and cultural characteristics. The Bayesian inference phylogenetic tree generated with Apn2/MAT intergenic spacer sequences of 59 isolates (one per orchard) revealed that C. alienum, C. asianum, C. fructicola, C. siamense, and C. tropicale were associated with symptoms of mango anthracnose. In this study, C. alienum, C. fructicola, C. siamense, and C. tropicale are reported for the first time in association with mango tissues in Mexico. This study represents the first report of C. alienum causing mango anthracnose worldwide. The distribution of Colletotrichum species varied among the mango growing states from Mexico. Chiapas was the only state in which all five species were found. Pathogenicity tests on mango fruit cultivar Manila showed that all Colletotrichum species from this study could induce anthracnose lesions. However, differences in virulence were evident among species. C. siamense and C. asianum were the most virulent, whereas C. alienum and C. fructicola were considered the least virulent species.

Changes in body composition in broilers by a sulfur amino acid deficiency during growth.

In the factorial approach, amino acid (AA) requirements are determined using the AA composition of retained protein, which is assumed to be constant. However, this hypothesis may not be valid because the AA composition of body protein can be affected by the diet. The objective of this study was to quantify the changes in chemical body composition of broilers receiving diets either deficient (TSAA-) or sufficient (TSAA+) in TSAA. Diet TSAA+ was formulated according to the Ross recommendation. Diet TSAA- provided 36% true digestible Met:Lys and 64% true digestible TSAA:Lys, which were, respectively, 34 and 22% lower compared with diet TSAA+. Performance and tissue weight gain between 7 and 42 d of age were not affected by the TSAA supply. In TSAA- chickens, protein gain was lower in the carcass (P < 0.01) and tended to be lower in the empty body (P = 0.06) and pectoralis major muscle (P = 0.10). Compared with TSAA+ chickens, lipid gain in TSAA- chickens was 78% greater in the pectoralis muscle (P < 0.001), 28% greater in abdominal fat (P < 0.05), and 10% greater in the carcass (P = 0.10). In the pectoralis muscle, there was a tendency for an increase in the redness value (a*; P = 0.10). The TSAA supply affected the AA composition of tissues and tissue gain, but the Met and Cys concentrations were changed only in the offal (P = 0.08). The deficient TSAA supply resulted in an increase in the Ser concentration in the empty body, carcass, and pectoralis muscle (P < 0.05). In contrast, it resulted in a decrease in the concentrations of Lys and Glu in the empty body, of Phe, Tyr, Gly, and Glu in the pectoralis muscle, and of Ala in the offal (P < 0.05). This indicates that although chickens cope with a TSAA deficiency predominantly by changing the protein and lipid concentration in the body, the AA composition is also affected. This calls into question the use of a constant ideal AA profile in poultry nutrition.

Direct analysis of aluminum alloys by CSigma laser-induced breakdown spectroscopy.

We report the application of CSigma laser-induced breakdown spectroscopy (Cσ-LIBS) to quantitative analysis of aluminum alloys without sample preparation. Cσ-LIBS simplifies strongly the conventional calibration procedure of LIBS, replacing it with a characterization stage performed from the spectrum of a single standard sample. The aim of this work has been to provide a complete evaluation of the use of Cσ-LIBS for direct analysis by obtaining its figures of merit, including precision and limits of detection. Ten elements (Si, Fe, Cu, Mn, Mg, Cr, Ni, Zn, Ti and Ca) are determined in a set of six certified samples with a wide range of concentrations, from percent down to µg/g levels. The average precision is 8.0% for concentrations higher than 0.1 wt% and 13% for concentrations between 0.1 wt% and 0.01 wt%. The limits of detection are in the range 1.4-9.7 µg/g.

Kinetic behavior of the reaction between hydroxyl radical and the SV40 minichromosome.

Aqueous solutions containing the minichromosomal form of the virus SV40 and the radical scavenger DMSO were subjected to gamma-irradiation, and the resulting formation of single strand breaks (SSB) was quantified. Under the irradiation conditions, most SSBs were produced as a consequence of hydroxyl radical ((•)OH) reactions. By controlling the competition between DMSO and the viral DNA substrate for (•)OH, we are able to estimate the rate coefficient for the reaction of (•)OH with the SV40 minichromosome. The results cannot be described adequately by homogeneous competition kinetics, but it is possible to describe the rate coefficient for the reaction as a function of the scavenging capacity of the solution. The experimentally determined rate coefficient lies in the range 1×10(9) - 2×10(9) L mol(-1) s(-1) at 10(7) s(-1), and increases with increasing scavenging capacity.

Repair of oxidative DNA damage by amino acids.

Guanyl radicals, the product of the removal of a single electron from guanine, are produced in DNA by the direct effect of ionizing radiation. We have produced guanyl radicals in DNA by using the single electron oxidizing agent (SCN)2-, itself derived from the indirect effect of ionizing radiation via thiocyanate scavenging of OH. We have examined the reactivity of guanyl radicals in plasmid DNA with the six most easily oxidized amino acids cysteine, cystine, histidine, methionine, tryptophan and tyrosine and also simple ester and amide derivatives of them. Cystine and histidine derivatives are unreactive. Cysteine, methionine, tyrosine and particularly tryptophan derivatives react to repair guanyl radicals in plasmid DNA with rate constants in the region of approximately 10(5), 10(5), 10(6) and 10(7) dm3 mol(-1) s(-1), respectively. The implication is that amino acid residues in DNA binding proteins such as histones might be able to repair by an electron transfer reaction the DNA damage produced by the direct effect of ionizing radiation or by other oxidative insults.

Uptake of WR-2721 derivatives by cells in culture: identification of the transported form of the drug.

When V79-171 cells are incubated in medium to which WR-1065 has been added the cells accumulate WR-1065 and disulfides of WR-1065 (WRSS) in a ratio of about 10:1. Analysis of the culture medium showed that it contained primarily WR-1065 but that significant levels of the symmetrical disulfide WR-33278 and of the mixed disulfide of WR-1065 with cysteine were also present. Since incubation of cells with either of the latter disulfides did not lead to uptake it was concluded that WR-1065 is the form of the drug taken up. The uptake rate on a per cell basis was shown to be independent of cell density, to be first order in the WR-1065 concentration in the incubation medium, to increase as [H+]-1.2 at medium pH values from pH 6.8 to 8.0, and to have a Q10 value (rate increase per 10 degrees C temperature increase) of 2.9 +/- 0.3 between 2 and 37 degrees C. Rates of WR-1065 uptake measured for HeLa, HT29/SP-1d, Me-180-VCII, Ovary 2008, and WI-38 cell lines were found to be similar to that measured for V79-171 cells. The results are consistent with uptake by nonmediated, passive diffusion of the uncharged form of WR-1065 across the plasma membrane but uptake mediated by a membrane transport system could not be rigorously excluded. Based upon these results and earlier findings it is postulated that the lower drug uptake seen in tumors as compared with normal tissues in animals treated with WR-2721 results from a combination of (a) slower conversion of WR-2721 to WR-1065 in tumors as a consequence of the lower inherent level of alkaline phosphatase and lower pH in tumors and (b) a decreased uptake rate of the WR-1065 present in tumors as a consequence of their lower pH.

Radioprotection of cells in culture by WR-2721 and derivatives: form of the drug responsible for protection.

Studies of V79-171 cells were undertaken to determine what extracellular or intracellular derivative of the drug WR-2721 is associated with radioprotection. The effect of preincubation at 23 +/- 1 degree C with WR-2721, and with derivatives of WR-2721 produced in medium containing alkaline phosphatase, upon survival of cells following subsequent gamma-irradiation was examined. It was established that WR-2721, WR-1065, WR-33278, WRSSCys, and other disulfide forms produced by reactions of WR-1065 with the medium do not provide significant protection when present only extracellularly. Protection was found to correlate with cellular levels of the thiol form of the drug (WR-1065) but not with the cellular level of the disulfide forms of WR-1065. Similar results were obtained with HeLa, Me-180, Ovary 2008, HT-29/SP-1d, and Colo 395 cell lines showing that human tumor cell lines behave in the same fashion as the V79-171 nontumorigenic hamster diploid cell line. None of the drug forms produced significant cytotoxicity under the conditions used. It was concluded that it is the cellular level of WR-1065 at the time of irradiation which determines protection. The results are consistent with protection mechanisms involving scavenging of hydroxyl radicals, hydrogen atom transfer to DNA radicals, depletion of oxygen near DNA, enhancement of rapid biochemical repair processes, or some combination of these mechanisms.

The level and source of free-methionine affect body composition and breast muscle traits in growing broilers.

Although dietary Met, as the first limiting amino acid (AA), has been extensively studied for poultry, little is known about how the supply and source of free Met affect tissue composition. The purpose of this study was to investigate the effect of feeding young broiler chickens with a deficient or sufficient TSAA (Met+Cys) supply, using either dl-Met (dl-Met+ and dl-Met-, for respectively diets sufficient and deficient in TSAA) or dl-2-hydroxy-4-methylthiobutyric acid (HMTBA+ and HMTBA-, for respectively diets sufficient and deficient in TSAA) as a Met source on tissue composition and breast muscle traits. For both Met sources, the deficient diets were formulated to provide true digestible Met:Lys and TSAA:Lys respectively 45% and 30% below that of the sufficient diets. Performance and tissue weights were affected by the Met supply but not by the Met source. In TSAA-deficient chickens, ADG and FCR, and protein content in empty body and pectoralis major muscles (PM) were lower than in TSAA-sufficient chickens (P < 0.05). Reducing the Met content of the diet increased the redness value of PM (a*) and the hue angle (H°; P < 0.01). The source of Met affected body AA composition and the partitioning of body Cys among tissues (P < 0.05). In TSAA-deficient birds, body Cys mass decreased in the commercial carcass and PM, but increased in the rest of the body (P < 0.01). The Met source also had an impact on the Cys mass, which was reduced in the commercial carcass and PM of dl-Met birds, but higher in the rest, especially in the feathers of TSAA-deficient birds (P < 0.05). The Met source, supply, or both altered the AA composition of the empty body, mostly in the commercial carcass. In conclusion, a dietary TSAA deficiency altered performance, tissue composition and quality traits of PM of broilers. There was no impact between dietary dl-Met and dl-HMTBA on performance or muscle weight, although the Met source affected the partitioning of Cys among tissues.

Synergism between the effects of dietary cholesterol and coconut oil on plasma, liver and lipoprotein composition of neonatal chick.

The nature of the synergism between dietary factors and the development of atherosclerosis has not been fully defined. Our studies showed that simultaneous supplementation of 10% saturated fat rich in 12:0 and 14:0 fatty acids (coconut oil) plus 1% cholesterol to the diet produced a sharp increase of plasma cholesterol, indicating a synergic influence of both dietary constituents. This increase was especially patent in the VLDL fraction, modifying the distribution of other lipid components between the core and the surface of these particles. These changes are consistent with the atherogenic function of VLDL and its responsiveness to dietary manipulation.

Variation of single-strand break yield with scavenger concentration for plasmid DNA irradiated in aqueous solution.

We have measured the yield of single-strand breaks (SSBs), induced by 137Cs gamma radiation as assayed by agarose gel electrophoresis, for three plasmids and SV40 DNA irradiated in aerobic aqueous solution. DNA SSBs are caused mainly by the hydroxyl radical under these conditions. To characterize the reactivity of DNA with the hydroxyl radical, we investigated the variation of the G value for SSBs [G(SSB)] with the concentration of added hydroxyl radical scavengers. We find that simple competition kinetics does not describe our results, but that a nonhomogeneous kinetics model is in good agreement. At a DNA concentration of 50 micrograms cm-3, G(SSB) for the direct effect is about 1 x 10(-5) mumol J-1 for the DNA substrates studied. This is equivalent to 2 x 10(-10) SSB Gy-1 Da-1. Estimates of the efficiency of SSB induction per OH. radical interaction with DNA (0.32-0.44) reveal that all plasmids are essentially equal in reactivity.

Variation of single-strand break yield with scavenger concentration for the SV40 minichromosome irradiated in aqueous solution.

We have measured the yield of single-strand breaks (SSBs) induced in aerobic aqueous solution by 137Cs gamma irradiation for the SV40 minichromosome as measured by agarose gel electrophoresis. Under these conditions, DNA SSBs are caused mainly by the hydroxyl radical. To characterize the reactivity of the SV40 minichromosome with the hydroxyl radical and to compare its behavior with that of naked DNA, we examined the variation of the G value for SSB formation, G(SSB), with the concentration of added hydroxyl radical scavengers. We find that simple competition kinetics is not applicable, but that a nonhomogeneous kinetics model is in much better agreement. Estimates of the efficiency of SSB induction per OH radical interaction with the SV40 minichromosome (0.04-0.05) indicate that this substrate is about five times more radioresistant than naked DNA at scavenging capacities < 10(8) s-1. At a DNA concentration of 50 micrograms ml-1, G(SSB) for the direct effect in the minichromosome is about 1 x 10(-5) mumol J-1 (2 x 10(-10) SSB Gy-1 Da-1), essentially equal to that for naked DNA.

The difference that linear energy transfer makes to precursors of DNA strand breaks.

Using agarose gel electrophoresis, we have measured the yields of DN A single- and double-strand breaks (SSBs and DSBs) for plasmid DNA irradiated in aerobic aqueous solution with either 137Cs gamma rays or 4He ions with a mean LET of 94 or 150 keV micron-1. The presence of dimethyl sulfoxide (DMSO) resulted in a decrease in the yields of both SSBs and DSBs, with a greater decrease being apparent for gamma irradiation than for 4He-ion irradiation. Irradiation by 4He ions in the presence of N-(2-thioethyl)- 1,3-diaminopropane (WR-1065) resulted in a decrease in the yield of SSBs and a slightly larger decrease in the yield of DSBs. Together with results obtained previously, these observations suggest a substantial contribution to the formation of SSBs and DSBs by 4He ions by species containing at least two radicals and more than two radicals, respectively.

Binding of radioprotective thiols and disulfides in Chinese hamster V79 cell nuclei.

Binding of thiols of varying charge (Z) in nuclei prepared in suspension was determined to assess the extent to which histones, Mg2+ spermine and chromatin structure influence counter-ion condensation of cationic thiols and co-ion depletion of anionic thiols at DNA. The nuclei were prepared in suspension buffer, washed and incubated in buffer containing thiol and graded amounts of Mg2+ and spermine. The nuclei were separated from the incubation medium by centrifugation through silicone oil, and the thiols were determined in the nuclear pellet and in the incubation buffer by labeling with monobromobimane and HPLC. Measurements of the water content of nuclei indicated that chromatin was fully condensed in buffer containing 5 mM MgCl2 and 115 mM KCl. Under these conditions nuclei incubated in 1 mM substrate had concentrations of 0.80 +/- 0.21 mM glutathione (Z = -1), 1.05 +/- 0.12 mM 2-mercaptoethanol (Z = 0), 0.95 +/- 0.15 mM cysteine (Z = 0), 0.75 +/- 0.29 mM cysteamine (Z = +1), 2.5 +/- 0.3 mM WR-1065 (Z = +2), 3.4 +/- 0.5 mM WR-35980 (Z = +3) and 12 +/- 2 mM WR-33278 (disulfide of WR-1065, Z = +4), respectively. Spermine up to 1 mM in the presence of 5 mM Mg2+ had little effect upon the binding of these thiols and disulfide, but did suppress the binding of 0.1 mM WR-33278, the results indicating that WR-33278 and spermine compete for the same sites with comparable affinity. From the results observed and the assumption that deviations from the bulk solution concentration (1 mM) result from counter-ion condensation within 3 nm of DNA, we estimate that WR-1065 (Z = +2), WR-35980 (Z = +3) and WR-33278 (Z = +4) were concentrated near DNA 6-, 8- and 20-fold, respectively, in the presence of histones, 5 mM Mg2+ and 1.0 mM spermine.

Characterization of the reaction rate coefficient of DNA with the hydroxyl radical.

Using agarose gel electrophoresis, we have measured the yield of single-strand breaks (SSBs) induced by 137Cs gamma irradiation in a variety of plasmid DNA substrates ranging in size from 2.7 kb to 38 kb irradiated in aerobic aqueous solution in the presence of the hydroxyl radical scavenger dimethyl sulfoxide (DMSO). Under these conditions DNA SSBs are caused mainly by the hydroxyl radical. Using the competition between DMSO and DNA for the hydroxyl radical, we have estimated the rate coefficient for the reaction of the hydroxyl radical with DNA. The results cannot be characterized by conventional steady-state competition kinetics. However, it is possible to describe the second-order rate constant for the reaction as a function of the scavenging capacity of the solution. The second-order rate constant increases with increasing scavenging capacity, rising from about 5 x 10(8) dm3 mol-1 s-1 at 10(5) s-1 to about 10(10) dm3 mol-1 s-1 at 10(10) s-1. This dependence of the second-order rate constant on the scavenging capacity appears to be more pronounced for larger plasmids.

The limits to radioprotection of Chinese hamster V79 cells by WR-1065 under aerobic conditions.

Clonogenic survival and drug content for Chinese hamster V79-171 cells incubated in suspension with WR-1065 prior to gamma irradiation have been determined. Factors that might influence the radioprotection by WR-1065 were investigated in control studies. Intracellular drug levels studied ranged between 0-36 nmol per 10(6) cells. In control studies, it was established that extracellular drug toxicity was not significant for cells in suspension at 10(6) per milliliter over short periods but was important when residual drug was present above 2 microM in the final plating of cells. Accumulation of intracellular drug above 30 nmol per 10(6) cells produced significant cytotoxicity in unirradiated cells. Irradiation with doses as high as 150 Gy produced no significant change in the total drug level or the thiol/disulfide ratio, either for the drug in the cells or for the drug in the medium. Preirradiation with 8 Gy did not change the ability of cells to import the drug but did appear to increase the cytotoxicity of the intracellular drug at levels above 25 nmol per 10(6) cells. There was no qualitative difference in the ability of WR-1065 to protect viable cells preirradiated with 8 Gy compared with protection of unirradiated cells. For a given gamma-ray dose from 2 to 40 Gy, there is a limiting value for surviving fraction which cannot be increased by further elevation of the intracellular drug level in V79-171 cells. Such limiting radioprotection was demonstrated for HT-29/SP-ld, HeLa, Me-180-VCII and OV-2008-VI human tumor cells.

Transport of aminothiol radioprotectors into mammalian cells: passive diffusion versus mediated uptake.

Water:n-octanol partition coefficients (KD) were determined for a series of radioprotective thiols to ascertain whether these could be used to estimate reliably their rates of uptake into mammalian cells by passive diffusion. Values of KD determined for thiols in 0.1 M potassium phosphate, pH 7.4, at 22 degrees C were: N-(2-mercaptoethyl)-1,3-diaminopropane (WR-1065, WRSH), 2.0 x 10(3); dithiothreitol, 1.4; 2-mercaptoethanol, 1.7; cysteamine, 180; 3-mercaptopropanoic acid, 450; mercaptosuccinic acid, 5 x 10(6) (extrapolated value). Predictions of uptake rates by passive diffusion into mammalian cells using these values and values for the membrane diffusion rate derived from empirical evaluation of appropriate values from the literature for erythrocyte permeability paralleled the experimental rates for WR-1065 and dithiothreitol but were about threefold lower. Although the utility of KD values for quantitative prediction of uptake rates is limited, the analysis clearly indicated that uptake of aminothiols having three or more ionized amino groups will not occur at useful rates by passive diffusion. Studies of WR-1065 import by Chinese hamster V79-171 cells at micromolar levels of WR-1065 revealed an uptake that could not be explained by passive diffusion. This uptake was not inhibited by substrates for common amino acid transport systems but was inhibited by polyamines and by 1 mM DTT, which suggested that WR-33278 (WRSSWR) formed by oxidation of WRSH was being transported by a polyamine transport system. This was confirmed by showing that WRSSWR is imported efficiently by V79-171 cells treated with D,L-2-difluoromethylornithine to deplete intracellular polyamines and hence enhance their transport. Spermine inhibited uptake of WRSSWR and WRSSWR inhibited uptake of [14C]spermine, confirming that a common system is involved in the uptake of these similar molecules, both having +4 charge. It was shown that after import WRSSWR is reduced to WRSH and that uptake at low micro-molar concentrations of WRSSWR results in marked cellular concentration of the drug. These results indicate that the spermidine/spermine transport system may also provide a feasible route for import of radioprotective aminothiols bearing net charges of +3 or +4 into mammalian cells.

Polyamine-induced compaction and aggregation of DNA--a major factor in radioprotection of chromatin under physiological conditions.

Spermine at physiological levels and ionic strength induces compaction and aggregation of SV40 DNA and minichromosomes resulting in marked radioprotection of the DNA against gamma-ray-induced formation of single-strand breaks. This phenomenon, termed the PICA effect, results in yields of single-strand breaks in DNA and minichromosomes comparable to those found with intact cells and is considered to be a major mechanism responsible for radioprotection of cellular DNA.

Effect of polyamine-induced compaction and aggregation of DNA on the formation of radiation-induced strand breaks: quantitative models for cellular radiation damage.

The yield of DNA single-strand breaks, G(SSB), upon gamma irradiation of SV40 DNA and SV40 minichromosomes in aqueous solution under aerobic conditions was determined at physiological ionic strength in the presence of various potential radioprotective agents. Putrescine (PUT), spermidine (SPD), glutathione, trans-4,5-dihydroxy-1,2-dithiane, 2-mercaptoethyl disulfide and cystamine, all at 0.1-10 mM, spermine (SPM, 0.1-1 mM) and WR-33278 (WRSSWR, 0.1-2 mM) lowered G(SSB) of SV40 DNA. These results were expected from the ability of these agents to scavenge OH radical in the bulk solution. However, SPD, above 10 mM, and SPM and WRSSWR, each above 2 mM, produced dramatic radioprotection attributed to polyamine-induced compaction and aggregation of the DNA (PICA effect). The DNA of SV40 minichromosomes was inherently less radiosensitive and was subject to a PICA effect at lower polyamine concentrations, i.e. approximately 5 mM SPD, approximately 0.6 mM SPM and approximately 0.5 mM WRSSWR. The PICA effect decreased G(SSB) for SV40 DNA and minichromosomes by one to two orders of magnitude, depending upon the scavenging capacity of the medium. The final yields were similar for SV40 DNA and minichromosomes and were comparable to the corresponding yield determined for cells. Results for the yield of double-strand breaks indicated that the yield of double-strand breaks, G(DSB), for DNA and minichromosomes is subject to a PICA effect by SPM and SPD comparable to that measured for G(SSB). Values of G(SSB) for SV40 DNA and minichromosomes subjected to the PICA effect were well approximated by calculations based upon a 30-nm cylinder assumed to model their condensed states. The results indicate that a major fraction of the formation of SSBs in condensed DNA and minichromosomes results from nonscavengeable radical intermediates. Minichromosomes subjected to the PICA effect of 2 mM SPM were protected against formation of radiation-induced SSBs 1.5-fold by 20 mM DTT but 5-fold by 10 mM DTT plus 10 mM WR-1065 relative to 2 mM SPM alone. Thus WR-1065 is capable of providing marked protection of compacted and aggregated minichromosomes, a protection ascribed to the chemical repair of DNA radicals by WR-1065.

Nuclear thiols: technical limitations on the determination of endogenous nuclear glutathione and the potential importance of sulfhydryl proteins.

Significant discrepancies were found between the values for glutathione levels determined by the Tietze enzymatic assay and those measured by labeling with monobromobimane followed by HPLC analysis when these methods were applied to proliferating and quiescent cells of the 66 murine mammary tumor line depleted of glutathione by buthionine sulfoximine or to nuclei prepared from these cells by permeabilization with Nonident detergent. The probable origin of the discrepancy was traced to the presence of acid-soluble sulfhydryl proteins in the extracts which are thought to lead to erroneous values in the Tietze assay method. Using the monobromobimane-HPLC method it was found that the low-molecular-weight thiol levels in nuclei prepared by detergent permeabilization equilibrate in less than 1 min with the permeabilizing medium, indicating that (i) endogenous nuclear glutathione levels cannot be determined reliably using conventional methods of cellular disruption and (ii) the endogenous nuclear glutathione level is likely to be the same as the cytoplasmic value. The levels of protein sulfhydryl associated with the nuclear preparations were found to be of the same magnitude as the cytoplasmic GSH level and must therefore be considered a potentially significant source of thiol capable of repairing DNA radicals.

Thiol uptake by Chinese hamster V79 cells and aerobic radioprotection as a function of the net charge on the thiol.

A series of thiols having net charge (Z) varying from -2 to +3 were studied using aerobic suspensions of Chinese hamster V79-171 cells in pH 7.4 medium at 297 K to evaluate the rate of uptake by cells and the extent of radioprotection as a function of thiol concentration in cells. For measurement of cellular levels, cells were separated from medium by centrifugation through silicone oil and tritiated water was employed to determine cell water volume. Estimated half-lives for uptake were: 2-mercaptosuccinate (Z = -2), greater than or equal to 1 h; 3-mercaptopropanoate (MPA, Z = -1), less than 2 min; 2-mercaptoethanol (2ME, Z = 0), less than 2 min; cysteamine (CyA, Z = +1), less than 2 min; N-(2-mercaptoethyl)-1,3-diaminopropane (WR-1065, Z approximately +2), approximately 40 min; N1-(2-mercaptoethyl)spermidine (WR-35980, Z approximately +3), greater than or equal to 10 h. After equilibration the cellular concentration of MPA was 60 +/- 8% of the medium level; the corresponding values for 2ME and CyA were 95 +/- 3 and 180 +/- 12%, respectively, but equilibrium was not reached for the other thiols studied. Those thiols taken up at significant rates were evaluated in terms of their ability to protect against aerobic gamma-ray-induced lethality. The results, summarized in terms of the cellular concentration of thiol (mmol dm-3) needed to achieve an aerobic radioprotection factor of 1.5, were as follows: MPA, 80 +/- 15; 2ME, 24 +/- 2; CyA, 4.7 +/- 1.3; WR-1065, 3.4 +/- 0.6. These values accorded well with those predicted from hydroxyl radical scavenging and DNA radical repair rates obtained using pBR322 DNA as a model system. This shows that hydroxyl radical scavenging and DNA radical repair are important mechanisms in the protection of cells by thiols and that the net charge on the thiol is a significant factor in its effectiveness. The results indicate that in air hydroxyl radical scavenging is the dominant mode of action by MPA, but that chemical repair of DNA radicals becomes significant for 2ME and is the dominant mechanism of protection for CyA and WR-1065.