Modelling nitrogen retention in floodplains with different degrees of degradation for three large rivers in Germany.

Floodplains perform a variety of ecosystem functions and services - more than many other ecosystems. One of these ecosystem services is the reduction in nitrogen (N) loads and a subsequent improvement to the water quality. Since diffuse and also point nitrogen sources continue to cause a variety of problems in rivers and floodplains, inundated floodplains could act as net sinks for N and are therefore of great importance throughout Germany and Europe. This study analyses the effects of riparian floodplains on N-retention on the landscape scale for three large river systems with different degrees of degradation. Two approaches, differing in terms of the complexity of their respective input data and methods, were applied under wet and dry conditions. Whereas the proxy-based approach considers proxy values for N-retention, the model-based approach accounts for event-driven dynamic input data such as the extent of the inundated floodplain and incoming loads. Comparing the results of the two approaches it can be observed that floodplains of the near-natural river can retain up to 4% of the river load under wet conditions. During such conditions N-retention in floodplains is similar to that of rivers. For the two other floodplains, the results of the two approaches were quite different, showing lower N-retention capacities. However, for these floodplains as well, both approaches are suitable for calculating measurable N-retention rates, which is an important result because it also suggests that even degraded floodplains still preserve this particular ecosystem function and therefore still contribute to improving the quality of river water.

Biological responses to extreme weather events are detectable but difficult to formally attribute to anthropogenic climate change.

As the frequency and intensity of extreme events such as droughts, heatwaves and floods have increased over recent decades, more extreme biological responses are being reported, and there is widespread interest in attributing such responses to anthropogenic climate change. However, the formal detection and attribution of biological responses to climate change is associated with many challenges. We illustrate these challenges with data from the Elbe River floodplain, Germany. Using community turnover and stability indices, we show that responses in plant, carabid and mollusc communities are detectable following extreme events. Community composition and species dominance changed following the extreme flood and summer heatwave of 2002/2003 (all taxa); the 2006 flood and heatwave (molluscs); and after the recurring floods and heatwave of 2010 and the 2013 flood (plants). Nevertheless, our ability to attribute these responses to anthropogenic climate change is limited by high natural variability in climate and biological data; lack of long-term data and replication, and the effects of multiple events. Without better understanding of the mechanisms behind change and the interactions, feedbacks and potentially lagged responses, multiple-driver attribution is unlikely. We discuss whether formal detection and/or attribution is necessary and suggest ways in which understanding of biological responses to extreme events could progress.

Sensitization to hyperthermia below 43 degrees C induced in Chinese hamster ovary cells by step-down heating.

Step-down heating (SDH) of Chinese hamster ovary cells consisted of an acute heat treatment at an elevated temperature followed immediately by chronic exposure to a lower hyperthermic temperature [step-down temperature (SDT)]. The survival curve at the SDT showed a reduced D0 (dose required to reduce the cell population to 37% on exponential part of the curve) relative to that of control cells only for SDT of 43 degrees C or less. The ratio r of D0's, D0 (control)/D0 (SDH), varied inversely with the SDT (e.g., r = 13 for SDT = 40 degrees C, whereas r = 1.3 for SDT = 43 degrees C). The degree of sensitization, as measured by r, was independent of the conditioning temperature (CT) and the conditioning time, provided that the CT was above 43 degrees C and the conditioning treatment reduced cell survival to 60% or less. The ratio r could be defined mathematically as a function of the activation energies for heat-induced cell killing above and below 43 degrees C, the SDT, and the CT. Biphasic heat survival curves, which appeared with heating at constant temperature below 43 degrees C, also appeared under SDH conditions, which suggested that thermotolerance, as represented by the resistant phase of the biphasic survival curve, was not affected by SDH. Fractionation experiments to measure the loss of interaction of heat damage remaining from the conditioning treatment with the low-temperature heat damage showed that 63% of the 45 degrees C sublethal damage was removed by 67 minutes at a constant exponential rate.

Thermotolerance in the murine jejunum.

The heat sensitivity of the murine jejunum from C3H/HeJ mice and its capacity to develop thermotolerance were measured. Exteriorized loops of murine jejunum were heated in water baths at temperatures of 43-46 degrees C for various times. Animal mortality was used to calculate the median lethal dose by day 7 (LD50/7) in minutes as a function of temperature. The LD50/7 value was independent of the length of the intestinal loop, the presence of serum in the heating medium, prior starvation of animals, and fluid replacement post heating. The time-temperature plot for the LD50/7 values had an exponential slope constant of -0.71 degrees C-1, similar to that for other normal tissues. A heat treatment of 5 minutes at 45 degrees C increased the LD50/7 (total time at 45 degrees C) from 5.9 minutes to 13.0 and 15.6 minutes for 1- and 3-day fractionation intervals, respectively. Death after intestinal heating was associated with septicemia and reached a peak on days 2 and 3 post hyperthermia. Gentamicin (0.1 mg/mouse/day) increased the LD50/7 at 45 degrees C from 5.9 to 9.6; however, septicemia was still noted in dying, gentamicin-treated mice. Additional antibiotics did not further increase the LD50/7. Heat fractionation with gentamicin increased the LD50/7 (total time at 45 degrees C) to 23.0 and 12.5 minutes at 45 degrees C for 1- and 3-day intervals, respectively. These data suggest that thermotolerance plays a significant role in the intestinal heat response, but that the heat damage to intestinal barriers against bacterial septicemia may be superimposed on the cellular damage to the crypt-villus system.

DNA degradation in chinese hamster ovary cells after exposure to hyperthermia.

Chinese hamster ovary cells grown in suspension showed a progressive reduction in the size of their nuclear DNA to 50 to 60S fragments after hyperthermia (43-48 degrees). This DNA degradation was not a homogeneous response but was observed only in cells incapable of attaching to a substratum after acute heating. The DNA degradation was associated with the inability of cells to exclude the vital stain, trypan blue. The degradation process appeared to be a result of nucleolytic enzyme digestion which accompanies cell necrosis. A similar phenomenon was observed in heated monolayer cells but only after significantly greater time-temperature exposures. Our results show that cellular subpopulations can be separated after hyperthermia and that these subpopulations are biochemically distinct and characterized by different viability.

Heat protection by glycerol in vitro.

Heating of either Chinese hamster ovary or HeLa cells in medium containing glycerol protected against thermal killing. Above glycerol concentrations of 100 mM, protection of Chinese hamster ovary cells increased in a concentration-dependent manner. Exposure to glycerol only, before or after heating at 45 degrees, did not protect against cell killing. Glycerol protection against thermal damage was also expressed at the subcellular level. The fractional increase in the protein:DNA ratio for nuclei from heated HeLa cells (15 min, 48 degrees) was 1.6 with heating in 1 M glycerol, compared to 2.0 for medium controls. Both glycerol (1 M) and heat-induced thermotolerance (4 hr, 41.5 degrees) partially reversed the sensitizing effects of pH 6.4 and stepdown heating at 41.5 degrees. The partial deprivation of nutrients achieved by incubating cells in Hanks' balanced salt solution-sensitized Chinese hamster ovary cells against 45 degrees hyperthermia. Glycerol reversed this sensitization but only when nutrient deprivation was short term (75 min). With a long-term, 8.5-hr exposure to glycerol in Hanks' balanced salt solution, cells were significantly more sensitive to heat killing at 41.5 degrees than were cells heated for an equal period in Hanks' balanced salt solution alone. The similarities in the characteristics of glycerol protection and heat-induced thermotolerance suggest a common mechanistic basis for the two phenomena, although the ability of glycerol to act as a sensitizer in nutrient-deprived cells is not understood.

Heat fractionation and thermotolerance: a review.

A rational approach to the design of clinical protocols combining fractionated hyperthermia plus X-irradiation or hyperthermia plus chemotherapy requires an understanding of the biology of fractionated heat alone. Mammalian cells growing in vitro can dramatically increase their tolerance to thermal damage (i.e., reduce the cellular inactivation rate) after prior heat conditioning. Although the mechanism(s) for this cellular thermotolerance is still unknown, it is apparent that the thermal history, the heat fractionation interval, and the recovery conditions all modify significantly the degree of thermotolerance subsequently exhibited. At the tissue level, the role of cellular thermotolerance is further complicated by host physiological mechanisms. Few data are available on heat fractionation in vivo, and the relative importance of physiological versus cellular effects remains to be defined.

Effect of hyperthermia on activity of three glycosyltransferases in Chinese hamster ovary cells.

We measured activities of three glycosyltransferases at various times during heat-induced thermotolerance development. Glycosyltransferases are normally located in the Golgi apparatus and catalyze cellular glycosylation reactions. UDP-Gal:N-acetylglucosamine beta 1,4-galactosyltransferase (beta 1,4-GalT) is known to participate in the formation of N-linked glycoproteins; when compared to cell survival, beta 1,4-GalT activity was significantly more heat resistant (50% loss of activity: 80 min, 45 degrees C) and showed little elevation at a time when thermotolerance was fully expressed. However, beta 1,4-GalT activity increased twofold by 24-h postheating when thermotolerance had begun to decay. Activity of beta 1,4-GalT was compared with glycosyltransferase activities that are considered to be specific for O-linked glycoproteins: UDP-Gal:N-acetylgalactosamine-beta 1,3-galactosyltransferase (beta 1,3-GalT), and UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (Gal-NAcT). Heat-inactivation experiments with heating times up to 60 min at 45 degrees C failed to reduce either activity below that of unheated control cells. Instead both beta 1,3-GalT and GalNAcT activity increased approximately twofold immediately after 10 min at 45 degrees C. Activity of beta 1,3-GalT rapidly decreased with time after heating and returned to control levels by 6-h postheating. In contrast, GalNAcT activity continued to increase with time after 10 min at 45 degrees C, and was 4.5-fold above unheated controls by 6-h postheating. GalNAcT activity returned to control levels 24- to 48-h postheating. A comparison with the cellular survival response showed that GalNAcT activity preceded thermotolerance expression by 2-4 h and also decayed more rapidly than heat resistance in thermotolerant cells. These data, together with other published results, suggest that expression of thermotolerance may be associated with enhanced glycosylation of intracellular proteins.

Protection against heat-induced cell killing by polyols in vitro.

The polyols erythritol and adonitol reduced 45 degrees heat killing in asynchronous Chinese hamster ovary cells. Heat protection by glycerol and erythritol increased with the apparent intracellular concentration, as inferred from cell volume measurements, and the number of hydroxyl groups per alcohol molecule. The nonlinear tetrahydroxy alcohol pentaerythritol did not protect but sensitized to heat killing. On cell survival curves, the reduced cell killing of protected cells was expressed by an increased Do for the pentahydroxy alcohol adonitol (0.3 M), whereas equimolar concentrations of glycerol increased primarily the Dq (quasithreshold dose) with little increase in Do. The distribution of Chinese hamster ovary cells within the cell cycle was unaffected by the presence of 0.3 M glycerol in the culture medium. However, the polyols erythritol and sorbitol caused a small but significant loss of cells from the heat-resistant G1 compartment. The cell cycle redistribution with prolonged incubation (6 hr) in polyol-supplemented medium is expected to increase the heat sensitivity of the perturbed cell population; the observed heat protection by polyols suggests that heat resistance in the presence of polyols is not an artifact of an asynchronous cell system. Instead, the data identify a family of heat-protective compounds that may occur naturally in mammalian cells.

Mechanism of spermidine cytotoxicity at 37 degrees C and 43 degrees C in Chinese hamster ovary cells.

Spermidine (SPD) cytotoxicity is mediated largely through SPD oxidation by serum amine oxidases at both 37 degrees C and 43 degrees C. Alkaline sucrose gradient data suggest random induction of DNA strand breaks. A dose response with time at 37 degrees C or 43 degrees C (constant SPD) or with SPD concentration (constant time at 43 degrees C) was observed in terms of both DNA strand breaks and cell survival. The generation of peroxide was demonstrated in the absence of cells by the addition of SPD to medium containing 10% fetal bovine serum. Toxicity of SPD was reduced in saline, with catalase-thiourea and aminoguanidine, and enhanced by prior depletion of glutathione. Thermotolerance induced 16 h previously did not protect against SPD toxicity, suggesting that the reactive species from spermidine oxidation and heat damage do not produce related subcellular lesions.

Protection against thermal cell death in Chinese hamster ovary cells by glucose, galactose, or mannose.

The addition of D-glucose, D-galactose, or D-mannose to culture medium increased survival of heated Chinese hamster ovary cells in a concentration- and time-dependent manner. Heat protection by sugars was not immediate but required prior incubation in the sugar medium before hyperthermia. The degree of heat protection conferred by each sugar and its time dependence differed characteristically: galactose protection appeared rapidly (within 1 hr) and was proportional to the galactose concentration in the medium up to 0.3 M. Glucose and mannose were less effective heat protectors at 0.3 M concentrations when compared with galactose, but cell survival after 40 min, 45 degrees, at concentrations below 0.1 M was similar in the three hyperosmotic sugar media. Heat protection by 0.3 M glucose under hyperosmotic conditions (600 mOSM) became apparent only after a preincubation period of at least 5 hr, 37 degrees. Under isoosmotic conditions (300 mOSM, 10% medium-10 mM morpholinopropanesulfonic acid-250 mM glucose), heat protection by glucose appeared more rapidly (3 hr), but the time dependence of heat protection was not eliminated. Under the same isoosmotic conditions in galactose medium, the survival of heated cells was not measurable. The 45 degrees survival curve in hyperosmotic galactose medium (6 hr, 37 degrees prior to heating, 0.3 M) was characterized by a DO of 10 min (controls, 3 min) and a quasithreshold dose of 17 min (controls, 17.5 min). When galactose-loaded cells were returned to fresh medium, heat protection decayed rapidly; cell survival measured 1 or 6 hr later showed a small degree of residual heat protection. A 5-hr incubation period at 37 degrees in galactose-supplemented medium resulted in a major intracellular accumulation of free galactose with smaller accumulations of glucose, sorbitol, and dulcitol. A similar incubation in glucose medium showed only minor intracellular elevations of polyols or sugars. A flow-cytometric analysis of the age distribution showed that incubation in the sugar-supplemented media slightly reduced the fractional cell population in G1 with concomitant gains in both the S- and G2-phase populations. Thus, heat protection by galactose or glucose is probably neither the result of a redistribution of cells in the cycle, nor does it always require the intracellular accumulation of free sugars and polyols. The greater degree of heat protection by galactose and its rapid manifestation under hyperosmotic conditions may be related to the intracellular accumulation of free galactose and/or its metabolites.

Cytotoxicity of hyperthermia combined with bleomycin or cis-platinum in cultured RIF cells: modification by thermotolerance and by polyhydroxy compounds.

The effect of thermotolerance and of polyhydroxy compounds on the cytotoxicity of bleomycin and cis-platinum was studied in cultured RIF tumor cells. Cell survival in response to drug-heat treatments was compared in cells not previously exposed to hyperthermia and in preheated cells that had developed thermotolerance. Since cellular accumulation of polyhydroxy compounds is a potential mechanistic basis of thermotolerance, we also compared cell survival of thermotolerant cells and chemically heat-protected cells. The cytotoxicity of bleomycin and cis-platinum in control cells treated with drug plus heat (43 degrees C, 1 h) was increased synergistically over the cytotoxicity of drug and heat alone. In thermotolerant cells, the synergistic interaction was largely reversed with the bleomycin-heat combination but retained with cis-platinum at 43 degrees C. In the absence of heat, bleomycin and cis-platinum showed similar cytotoxicity in control and thermotolerant cells. The addition of heat protectors (erythritol or galactose) modified the drug-heat cytotoxicity similar to thermotolerance. The synergistic interaction of bleomycin-43 degrees C, but not cis-platinum-43 degrees C, was reversed by the polyhydroxy compounds.

Characterization of a cisplatin-resistant subline of murine RIF-1 cells and reversal of drug resistance by hyperthermia.

The development of tumor cell drug resistance is a major obstacle which often leads to failure of cancer chemotherapy. Therefore, reversing the cell drug resistance would have important implications in cancer treatment. We have developed a cisplatin-resistant mouse tumor cell line from the radiation induced fibrosarcoma (RIF-1) parental line; this line is named RIF/ptr1 versus the parental line RIF/pts1. It is shown that the formation of cisplatin-DNA interstrand cross-links is the same for both cell lines although the intracellular cisplatin concentrations of resistant line is significantly lower. The cytosolic activities of glutathione reductase, glutathione peroxidase, and DT-diaphorase were the same in two cell lines. However, the concentration of glutathione was significantly higher in the resistant line. The resistant line was shown to be more sensitive to the cytotoxicity of heat (43 degrees C) but the combination of heat and drug had the same tumoricidal effect for both cell lines. The addition of verapamil also had a similar effect on both cell lines. We conclude that the major difference between these two lines was the glutathione-related detoxification of platinum. Regardless of drug resistance, the combination of drug and heat can effectively kill both cell lines. Elevated glutathione in RIF/ptr1 cells may be associated both with enhanced heat sensitivity and drug resistance such that combined treatments with drug and heat were equally effective in killing cells of either line.

Effect of step-down heating on the cytotoxicity of adriamycin, bleomycin, and cis-diamminedichloroplatinum.

The cytotoxicity of a number of anticancer drugs can be potentiated by mild hyperthermia. Since the cytotoxicity of low-hyperthermic temperatures in the range of 39-43 degrees can be potentiated by using a step-down heating (SDH) sequence (short times at greater than or equal to 45 degrees followed by longer times at lower temperatures), we investigated the combined effects of SDH (specifically 45 degrees, 10 min, followed by time at 41.5 degrees) and three chemotherapeutic drugs known to be more toxic at elevated temperatures. In agreement with previous observations with Chinese hamster cells, we found that cultured RIF tumor cells showed an increased rate of cell killing at 41.5 degrees when the low-temperature heat treatment was preceded by an acute heat shock of 10 min, 45 degrees. Similarly, RIF cells showed enhanced cytotoxicity of Adriamycin, bleomycin, and cisplatin at 41.5 degrees over that at 37 degrees, as reported previously for hamster cells. When SDH and drug exposures were combined, the rate of cell killing was greater than that observed with the drugs at a constant 41.5 degrees. SDH alone reduced the Do on the 41.5 degrees survival curve from approximately 500 min to 100 min after 10 min at 45 degrees. Adriamycin cytotoxicity (0.3 microgram/ml) at 41.5 degrees was characterized by a Do of approximately 160 min for treatment times of 1 to 4 hr; with SDH, this was reduced to 52 min. Similarly, bleomycin (5 micrograms/ml) and cisplatin (0.5 microgram/ml) showed a decrease in Do by factors of 1.4 (68 to 48 min) and 2 (25.2 to 13.5 min), respectively, with the SDH sequence when compared with drug toxicity at 41.5 degrees alone. However, the sensitization to cisplatin was the same when 10 min at 45 degrees was followed by drug exposure at 37 degrees, or at 41.5 degrees. The results indicate that Adriamycin and bleomycin, but not cisplatin, cytotoxicity was increased by SDH over that achievable with 41.5 degrees alone. Possible clinical implications are discussed briefly.

Tumor-targeted delivery of 8-hydroxyquinoline.

RIF-1 mouse tumors express high levels of beta-glucuronidase activity relative to most normal tissues. The high activity can be exploited for targeting specific drugs preferentially to tumor tissues. In this study we examined the kinetics of 8-hydroxyquinoline (8-OHQ) accumulation in tumor and in several normal tissues resulting from the in vivo deconjugation of 8-hydroxyquinolyl-glucuronide (8-OHQ-GlcA). Tumors were acidified with D-glucose and NaHCO3 prior to the administration of 8-OHQ-GlcA; subsequently the deconjugated aglycone, 8-OHQ, accumulated preferentially in tumors and reached peak levels between 30 and 60 min after the 8-OHQ-GlcA injection. Mild hyperthermia of 30 min at 43 degrees C to the tumors further increased their peak 8-OHQ levels by a factor of 2-3. Some normal tissues, mostly kidney, liver, and colon, also accumulated 8-OHQ, but the aglycone appeared early in the normal tissues (near 30 min post-injection) and was significantly reduced by 60 min when 8-OHQ remained high in the tumor. Administration of 8-OHQ-GlcA alone, without prior tumor acidification, failed to produce measurable accumulations of 8-OHQ in tumors and in normal tissues. Tissue clearance of 8-OHQ is mediated primarily by the enzymatic reconjugation of 8-OHQ via UDP-glucuronosyltransferase (UDPGT). UDPGT activity was high in liver, kidney, and bowel, but low in the RIF tumor, spleen, muscle, and brain. Hyperthermia had only a modest effects on UDPGT activity: a heat dose of 30 min at 45 degrees C reduced activity less than 60%. Thus, preferential accumulation and prolonged retention of 8-OHQ in RIF tumors may be caused by a combination of factors: a) high tumor beta-glucuronidase activity, b) selective tumor acidification during hyperglycemia, c) low tumor UDPGT activity, and d) other factors, such as tumor blood flow.

Interactions of BCNU, low pH, glucose and hyperthermia in cultured RIF cells.

The interactions of BCNU (1,3-bis[2-chloroethyl]-1-nitrosourea) with low pH, glucose and hyperthermia were studied in cultured RIF tumor cells. The effect of a mild heat treatment of 43 degrees C, 1 h at pH 7.4 on cell killing [surviving fraction (S) = 0.27 +/- 0.05, standard error of the mean (S.E.)] was significantly enhanced by pH 6.5 (S = 0.11 +/- 0.02, S.E.) and 50 mM D-glucose (S = 0.14 +/- 0.01, S.E.). When heat (43 degrees C, 1 h) was added to BCNU, cytotoxicity was increased approximately 14-fold over BCNU alone. Moreover, pH 6.5 increased killing with BCNU and heat by an additional factor of 28. The presence of glucose at 37 degrees C at either pH 6.5 or 7.4 reduced BCNU toxicity in a dose dependent fashion. However, the presence of glucose did not reduce cell killing by BCNU at 43 degrees C. As a result BCNU cytotoxicity was enhanced by approximately 2 orders of magnitude when tumor cell acidification (glucose and low pH) was combined with BCNU and heat.

Partial characterization of a Cisplatin-resistant subline of murine rif-1 tumor-cells.

We have developed a drug-resistant cell line (RIF/Ptr1) (R) from the murine radiation-induced fibrosarcoma (RIF-1) also designated Pts or (S). This subline has been characterized previously by an increased resistance to cisplatin (cis-diamminedichloroplatinum(II) or CDDP), lowered intracellular CDDP concentrations, and elevated intracellular glutathione (GSH) levels (1.4-2.5-fold) but unaltered formation of CDDP-DNA interstrand cross-links. In this work, we have shown that RIF/Ptr1 cells were also resistant to carmustine (BCNU) and X-irradiation. Neither cell line had P-glycoprotein 170. The intrastrand CDDP-DNA adduct level was proportional to the concentration of intracellular CDDP. The oxygen consumption, ATP level, and glycolysis were similar in both cell lines. The cisplatin influx and efflux showed that the RIF/Ptr1 cells had lower drug influx and higher drug efflux compared to RIF-1. We conclude that the major difference between the cisplatin sensitive and cisplatin-resistant cells in this model is the regulation of cisplatin transport probably at the cell membrane level suggesting that a membrane active transport system other than P-glycoprotein 170 is involved. Whether glutathione is linked to the putative membrane transporter needs further investigation.

Temperature-dependent induction of thermotolerance by ethanol.

Ethanol (1 M) cytotoxicity in asynchronous Chinese hamster ovary cells was strongly temperature dependent, yielding families of cell survival curves between 34 and 39 degrees C that were similar to those obtained at hyperthermic temperatures in medium without ethanol. Below 36 degrees C, survival curves were biphasic, indicating the development of thermotolerance during ethanol exposures. At room temperature (22 degrees C) ethanol was completely nontoxic with incubation periods up to 6 h. A comparison of survival curves with and without ethanol showed that the major effect of ethanol was an effective temperature shift of circa 6.5 degrees C, i.e., the cell survival curve at 37 degrees C in 1 M ethanol was equivalent to that at 43.6 degrees C in medium without ethanol. In addition to the effective temperature shift, ethanol also resulted in sensitization to "heat" with a temperature dependence that was similar to the stepdown heating effect. When thermotolerance was induced with acute ethanol exposures (25 min, 37 degrees C or 60 min, 35.5 degrees C), the kinetics and the magnitude of tolerance were similar to those after isotoxic conditioning treatments with heat alone (10 min, 45 degrees C). In contrast, equimolar ethanol at 22 degrees C did not induce thermotolerance. These data provide a rationale for conflicting results in the literature regarding thermotolerance induction by ethanol. Both heat sensitization and the induction of thermotolerance are interpreted as the effect of ethanol on the solution properties of intracellular water. These solvent alterations reduce the temperature necessary to elicit cytotoxicity and the development of thermotolerance.

Expression of thermotolerance following microinjection of glutathione disulfide.

Asynchronous Chinese hamster ovary cells were microinjected with glutathione disulfide (GSSG). Successfully injected cells were scored by coinjecting FITC-dextran with GSSG, followed by fluorescent microscopy. After microinjection, cells were incubated for 2.5 h at 37 degrees C to permit thermotolerance development and then heated at 45 degrees C for 40 min. Cellular heat sensitivity was quantitated by counting the number of grains per cell after labeling heated cells with tritiated amino acids and processing for autoradiography. The data show that microinjection of GSSG induced thermotolerance which increased the number of grains per cell up to 500% of controls. Cells that were exposed to similar concentrations of GSSG in culture medium without microinjection or microinjected without GSSG did not develop thermotolerance.

Tumor-targeted cell killing with 8-hydroxyquinolyl-glucuronide.

Many tumors show elevated levels of hydrolytic enzymes that may be associated with invasive processes. The RIF-1 murine tumor has levels of beta-glucuronidase that are more than four times higher than those in liver. Elevated tumor glucuronidase levels can be used as a basis for tumor-targeted therapy when systemically administered glucuronides of cytotoxic drugs are deconjugated preferentially at the tumor site. In this study we have used 8-hydroxyquinoline (8-OHQ) as a model compound for such a tumor-targeting concept. We showed that RIF tumors and spleen had the highest beta-glucuronidase activity in C3H mice; for example, RIF tumors released approximately seven times more phenolphthalein per gram of tissue from its glucuronide than liver, when compared under identical conditions. In vitro, low concentrations of 8-OHQ that might be achievable in vivo, ranging from 1 to 10 microM reduced cell survival by four orders of magnitude, while 1 mM 8-hydroxyquinolyl-glucuronide (1 h, 37 degrees C) resulted in only modest (S = 54%) cytotoxicity. Combination treatments of 8-OHQ (2.5 or 5 microM) with either hyperthermia or X radiation did not significantly change the slope of survival curves for RIF tumors in vitro, but suggest that targeted 8-OHQ toxicity combined with local hyperthermia and/or irradiation may be useful for significantly increasing therapeutic gains in vivo.