TNF, IL-1 and IL-6 in circulating blood after total-body and localized irradiation in rats.

The levels of TNF, IL-1 and IL-6 in circulating blood of female WAG/Rij rats were assessed both after total-body irradiation (TBI) and localized irradiation of the right hind leg. The results show that enhanced levels of IL-1 in the circulation reflect a stress situation presumably resulting from handling and halothane anesthesia of the animal. Neither localized irradiation nor TBI resulted in further enhanced levels of IL-1. Both TBI and localized irradiation, lead to a small but significant increase in IL-6 levels in serum from circulating blood. After TBI this increase dissipated rapidly, 24 h after TBI increased levels are not found. After localized irradiation IL-6 levels remain elevated for a longer period. Still two weeks after irradiation, the longest time investigated, increased levels were observed. We did not observe increased TNF levels after localized irradiation or after TBI.

Lack of thermal enhancement for taxanes in vitro.

The taxanes represent a new class of clinical chemotherapeutic agents. A series of in vitro studies were independently of each other initiated in two different institutes (Amsterdam and Madison) to test the hypothesis that hyperthermia might enhance the cytotoxicity of taxanes. Clonogenic capacity experiments (Amsterdam) included the exposure of R1- and SW 1573-cells to 1, 4, or 24 h of paclitaxel with heat 43 degrees C x 60 min in the last hour of drug treatment or at 24, 48 as well as 72 h post drug treatment. Survival assay experiments (Madison) included the exposure of L-929-cells to paclitaxel and docetaxel for 24 h with heat 41.8 degrees C x 60 min the first or last hour of drug treatment as well as 24 and 48 h post treatment. No thermal enhancement of cytotoxicity for the taxanes was observed in these human and murine cell lines, with congruent data in both institutes. In addition, high performance liquid chromatography studies at 41.8 degrees C and 43 degrees C demonstrated paclitaxel and docetaxel were heat stable.

Hyperthermia enhances the cytotoxicity and platinum-DNA adduct formation of lobaplatin and oxaliplatin in cultured SW 1573 cells.

The cytotoxicity of cisplatin and cisplatin-DNA adduct formation in vitro and in vivo is clearly enhanced by hyperthermia. We investigated whether cytotoxicity and platinum-DNA adduct formation of two promising new third-generation platinum derivatives, lobaplatin [1,2-diamminomethylcyclobutane platinum(II) lactate] and oxaliplatin [oxalato-1,2-diaminocyclohexane platinum(II)], are also enhanced by hyperthermia. Cisplatin was used for comparison. SW 1573 cells were incubated with cisplatin, lobaplatin or oxaliplatin at different concentrations for 1 h at 37 degrees, 41 degrees and 43 degrees C. The reproductive capacity of cells was determined by cloning experiments. Immunocytochemical detection of platinum-DNA adducts was performed with the rabbit antiserum NKI-A59. At 37 degrees C, cisplatin was the most cytotoxic, followed by oxaliplatin and lobaplatin. Hyperthermia clearly enhanced the cytotoxicity of cisplatin, lobaplatin and oxaliplatin. There was no further increase in cytotoxicity at 43 degrees C compared to 41 degrees C for cisplatin and oxaliplatin. A further increase in cytotoxicity at 43 degrees C was observed for lobaplatin. At 43 degrees C thermal enhancement was higher for lobaplatin than for oxaliplatin, with the reverse pattern at 41 degrees C. For both drugs, thermal enhancement of cytotoxicity was lower than observed for cisplatin. Immunocytochemical detection of platinum-DNA adducts was feasible for all the drugs. Adduct formation was enhanced at 43 degrees C for cisplatin, lobaplatin and oxaliplatin with a relative increase of 410%, 170% and 180%. These results seem to confirm that an increase in platinum-DNA adduct formation is involved in the in vitro thermal enhancement of cytotoxicity. The observed thermal enhancement of cytotoxicity of lobaplatin and oxaliplatin in vitro warrants further in vivo investigations.

Cytokine production after whole body and localized hyperthermia.

The levels of TNF, IL-1 and IL-6 in circulating blood female WAG/Ry rats were assessed in relation to treatment with localized hyperthermia of the right hind leg or with whole-body hyperthermia (WBH). After a localized treatment for 30 min at 43 or 44 degrees C no detectable increase in levels of IL-6 or TNF was obtained. Hyperthermia for 30 min at 45 degrees C led to an elevated level of IL-6 of 19.4 +/- 5.2 U/ml above the control level of 24 h after treatment. Levels of IL-1 were never higher than those in control animals that received only anaesthesia. Anaesthesia induced a peak level of approximately 131 U/ml IL-1 6 h after treatment. Serum levels of IL-1 and IL-6 are enhanced after WBH. IL-1 reaches a peak level already during WBH about 15 after reaching 41.5 degrees C. IL-6 levels were not enhanced during WBH but 1 h after WBH a clear peak was observed. Anaesthesia with sham WBH did not lead to enhanced IL-6 levels but enhanced IL-1 levels were clearly detected. We did not detect TNF in any sample after WBH. It is concluded from the present results that IL-6 is not induced by a 'standard' treatment of localized hyperthermia as used in oncotherapy (i.e. 60 min at 43 degrees C) to such a high level locally that this is reflected in increased levels in circulating blood. WBH at clinically relevant temperatures leads to enhanced levels of IL-1 and IL-6. The difference in IL-6 response after WBH or localized hyperthermia probably is related to the fact that in WBH also the bone marrow is treated. This may lead to stimulation of this important stem cell compartment of the peripheral blood. The sequence of appearance of IL-1 and IL-6 after hyperthermia is akin to the sequence in an inflammatory response. However, the experiments with sham treatment show that IL-1 may appear in the circulating blood not followed by IL-6. These results indicate that enhanced IL-1 levels may reflect a stress reaction of the animal related to the (sham) treatment. Enhanced levels of IL-1 after WBH correlate with enhanced levels of ACTH in the circulating blood.

Collagen content in rat liver after experimentally induced cholestasis followed by choledochojejunostomy and X-irradiation.

The right part of the median lobe of the liver of female Wistar rats was irradiated, 12.5 or 25 Gy, at a field size of 15 x 20 mm. The central part of the irradiated liver lobe was fixed and used for the estimation of the collagen protein ratio by means of the Sirius Red-Fast Green extraction method, immediately, 8, 16 or 32 weeks after irradiation. No significant increase in collagen content could be demonstrated in this time range, both after irradiation at 12.5 Gy and at 25 Gy. Partial hepatectomy according to Higgins led to rapid regrowth of the remaining liver lobes. The right lobe grew out rapidly to replace the median lobe. Two days after partial hepatectomy the right lobe was irradiated at the same field size. Measurement of the collagen protein ratio in this experiment did not show a significant increase 8, 16 or 32 weeks after irradiation. However, the 25 Gy group did not survive long enough to obtain data at 16 or 32 weeks. The animals in this latter experiment suffered from ascites before dying. Experimentally induced cholestasis was obtained by ligation and partial resection of the common bile duct. After two weeks of cholestasis the bile flow was restored by Roux-en-Y choledochojejunostomy. The effect of irradiation 2 days after repair surgery was studied. Without irradiation the collagen protein ratio is increased. Irradiation of the right part of the median lobe led to a relatively enhanced collagen content in this lobe. Our results indicate that radiation itself does not lead to a significantly enhanced degree of fibrosis in the liver. However when an increase in collagen content was induced by cholestasis, the partial "dilution" of enhanced fibrosis as a result of proliferation of liver parenchyma cells following repair surgery was inhibited by irradiation.

Effect of hyperthermia on the cytotoxicity of 2',2'-difluorodeoxycytidine (gemcitabine) in cultured SW1573 cells.

Difluorodeoxycytidine (dFdCyd, gemcitabine) was tested for cytotoxicity in cultured human lung-cancer cells SW1573 in combination with 1 hr hyperthermia at 43 degrees C. The results show that the timing is extremely important. Simultaneous application led to decreased cytotoxicity, whereas an interval of 20 or 24 hr between exposure to dFdCyd and hyperthermia led to enhanced cell killing. The decrease in cytotoxicity after simultaneous hyperthermia and dFdCyd probably results from inhibition of activation of dFdCyd to the triphosphate metabolite. The enhanced cytotoxicity in sequential application of dFdCyd and hyperthermia is not caused by accumulation of cells in a sensitive cell-cycle phase. Our results show that the G1 phase becomes relatively abundant 20 hr after exposure to 0.1 microM dFdCyd, approximately 48% versus 31% in control cultures. Presumably, inhibition by hyperthermia of repair of DNA damage plays a role. Our results confirm earlier data with regard to reutilization of activated dFdCyd at high cell density. dFdCyd was clearly more toxic to SW1573 cells at 4 x 10(5) cells per dish than at 400 cells per dish. This reutilization of activated drug is evidently not a restricted property of a particular cell line and may add to the value of the drug in cancer treatment.