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8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) is one of the mutagenic base modifications produced in DNA by the reaction of reactive oxygen species. The biological significance of 8-oxo-dG is shown by the existence of repair pathways that are able to recognize and remove this lesion from both DNA and the nucleotide pool. The final outcome of these evolutionarily conserved repair mechanisms in man is excretion of 8-oxo-dG/8-oxo-Gua from the intracellular to extracellular milieu including the blood plasma and urine. The aim of this investigation was to establish dose response relations for radiation-induced appearance of extracellular 8-oxo-dG in cellular model systems. Here we report on excretion of 8-oxo-dG after in vitro irradiation of whole blood and isolated lymphocytes with clinically relevant doses. We find that this excretion is dependent on dose and individual repair capacity, and that it saturates above doses of 0.5-1 Gy of gamma radiation. Our data also suggest that the nucleotide pool is a significant target that contributes to the levels of extracellular 8-oxo-dG; hence the mutagenic target for oxidative stress is not limited to the DNA molecule only. We conclude that extracellular 8-oxo-dG levels after in vitro irradiation have a potential to be used as a sensitive marker for oxidative stress.

Clinical (Dimery and Hong, J Nat Cancer Inst 1993; 85: 95- 111) and experimental studies (Scanlon et al., Proc Natl Acad Sci USA 1986; 83: 8923-5; Lewin et al., In Vivo 1990; 4: 277-82) have indicated an increased cytotoxic effect, when cisplatin (CDDP) is combined with 5-fluorouracil (5-FU). Addition of 5-FU abolishes the G2-arrest induced by CDDP (Lewin et al., In Vivo 1990; 4: 277-82; Nylén et al., Acta Oncol 1996; 35: 229 35). The mechanism for the synergy is unclear. Activation of p34cdc2 is necessary for progression from G2 to mitosis (Lewin et al., Anti-Cancer Drugs 1995; 6: 465-70). The aim was to study p34cdc2, cdc25C and weel after treatment of mammalian tumour cells in vivo with CDDP as single agent or in combination with 5-FU. CDDP prevented activation of p34cdc2 by keeping cdc25C inactive and weel active. Addition of 5-FU to CDDP decreased the expression of weel and promoted cdc25C-activation. p34cdc2 was dephosphorylated by cdc25C and activated. Alterations in activity of cdc25C and weel after drug combination were due to changes in the protein amount, rather than to changes in the phosphorylation degree.

The effects on incorporation into DNA of the deoxyribonucleotides dCTP and dTTP and the DNA synthesis rate after treatment with cisplatin (CDDP), 5-fluorouracil (5-FU) or a combination of CDDP and 5-FU were studied in ascites sarcoma (Bp8) growing in mice. Single administration of CDDP gave an early (1 h) transient increase in the DNA-synthesis followed by a decrease. 5-FU as single agent did increase the rate of DNA synthesis after 6 h with a maximum at 10 h. The combination of CDDP and 5-FU markedly increased the rate of DNA synthesis up to 6 h as compared to single drug treatment. Although the dCTP pool increased after combined treatment, while the dTTP pool was unchanged, no alterations in the proportions of dTTP and dCTP incorporated into DNA could be detected. Hence, misincorporation of pyrimidines is not the mechanism for the synergistic effect of the combination of CDDP and 5-FU.

The effect of increasing temperature up to 43 degrees C on thymidine (TdR) salvage, i.e. uptake of TdR, thymidine kinase (TK) activity, intracellular amount of thymidinetriphosphate (dTTP) and specific activity of 3H-dTTP and incorporation of dTTP into DNA, was studied in ascites tumour cells in vitro. While the uptake of [3H]TdR was almost unchanged up to 40.5 degrees C and 2.4 h followed by a decrease at later times and higher temperatures, TK activity declined already after 30 min in a temperature-dependent way, leading to a decrease in cellular amount of dTTP reaching almost undetectable values at 43 degrees C and 6.0 h. The specific activity of [3H]dTTP in the cell was elevated at 39 degrees C and 43 degrees C, but somewhat reduced at 40.5 degrees C. Incorporation into DNA of [3H]TdR decreased in a temperature-dependent way with increasing incubation time. After correction for specific activity of [3H]dTTP, however, the incorporation of [3H]TdR into DNA at 39 degrees C and 40.5 degrees C was unchanged up to 2.4 h and for 40.5 degrees C up to 6.0 h, while incorporation into DNA at 43 degrees C was still reduced. The overall protein synthesis was also reduced in a temperature-dependent way. We concluded that phosphorylation of TdR to dTMP was more sensitive to moderately elevated temperatures than uptake of TdR and DNA synthesis per se, caused by a reduced TK activity. The decline in TK activity was probably due to a decrease in TK polypeptide synthesis.

Inhibition of the salvage DNA synthesis was studied in ascites tumour cells up to 240 min at moderately increased temperatures of 39 degrees C, 40.5 degrees C and 43 degrees C alone or in combination with adriamycin (ADR). Hyperthermia and ADR acted additively on salvage DNA synthesis. The [3H]thymidine (TdR) incorporation was strongly related to the cellular uptake of [3H]TdR. In order to evaluate the significance of the decrease in [3H]TdR uptake for the incorporation into DNA, the thymidine kinase (TK) has also been studied. TK activity started to decrease at a temperature of 39 degrees C, probably due to inhibition of translation and/or transcription of the enzyme, and was almost completely inhibited at 43 degrees C. ADR did not affect TK activity. Inhibition of DNA synthesis by aphidicolin neither decreased the ability of the cell to take up [3H]TdR nor the ability to affect TK activity. We concluded that moderate increases in temperature alone, or in combination with ADR, inhibit salvage DNA synthesis by inhibition of TdR uptake, possibly due to inhibition of phosphorylation; ADR, on the contrary, inhibits TdR uptake by other mechanisms such as changes in membrane function.

The effect of 12, 24 and 36 mg/kg body weight doses of fluorouracil (5-FU) on the Bp 8 ascites sarcoma growing in vivo was studied. From sequential studies of the total number of cells together with the composition of cells in the cell cycle, the cell cycle flow was calculated and correlated to the pharmacokinetics, which was determined by using 3H-5-FU. The dose of 12 mg/kg 5-FU affected cell growth between 24 and 72 hours, while the effect of higher doses was immediate. An early block in outflow of cells from G1 was followed by an increased outflow, indicating an early inhibition followed by an enhancement of the initiation of the DNA synthesis. This increased outflow from G1 together with the decrease in outflow from the early S-phase, i.e. decreased DNA synthesis, resulted in an accumulation of cells in the early part of the S-phase. The prolonged effects on the cell growth and the cell cycle flow despite the very fast decline in the drug concentration both in the ascites fluid and within the cells, together with a constant level of the drug in the macromolecular fraction, suggest an interaction between 5-FU and RNA/DNA at later times rather than an inhibition of the thymidylate synthetase activity.

Cell populations obtained by bronchoalveolar lavage (BAL) from humans showed varying proportions of lymphocytes and macrophages, about 5% and 94%, respectively, in healthy controls (n = 12), and about 24% and 75%, respectively, in patients with sarcoidosis (n = 13). The present study was carried out to examine centrifugal elutriation as a means to obtain from human BAL fluids highly purified populations of viable cells, required for further biochemical investigations. After centrifugation of BAL fluids at 400 g for 5 min, cells were resuspended in Hank-Tris solution, loaded into a Beckman elutriator rotor (rotor speed 2800 rpm) and elutriated with Hank-Tris in approximately 10 min. Cells were collected at flow rates of 20 ml/min (fraction I), 32 ml/min (fraction II) and, after stopping the rotor, 80 ml/min (fraction III). Fraction I contained mainly lymphocytes, about 54% and 74% in controls and sarcoidosis, respectively. Fraction III consisted almost solely of macrophages, while fraction II contained mixed populations. Total cell recovery was about 74% and 55% in controls and sarcoidosis, respectively. Half of the missing cells were lost during centrifugation, the rest during elutriation. Cell viability was 85-90% after the first centrifugation. Following elutriation, the viability was found unchanged in fraction III, but decreased to about 50% in fraction I of controls. The decrease may be due to the accumulation of dead cells with decreased density in this fraction. The results showed that centrifugal elutriation is a highly suitable technique for the isolation of macrophages from human BAL fluids. Enriched lymphocyte populations may be obtained from sarcoid BAL fluids.

The cellular ATP content and the phosphorylation potential, defined as the ATP, ADP and inorganic phosphate (Pi) ratios, of exponentially growing Ehrlich ascites tumour cells were compared with cells at the plateau phase of growth. These phosphorus compounds were measured using 31P-NMR-spectroscopy immediately after removal of the cell material from the host and in their ascites fluid reflecting in vivo growth conditions. Reaching the plateau phase of growth, the ATP content and the phosphorylation potential decreased. Upon addition of glucose, the phosphorylation potential immediately increased. We concluded that the reduced phosphorylation potential was due to a limited availability of glucose in spite of the nearly normal blood glucose concentration found. An increasing diffusion distance from the host to all parts of the tumor is a possible reason for that.