In vitro and in vivo genotoxicity of radiofrequency fields.

There has been growing concern about the possibility of adverse health effects resulting from exposure to radiofrequency radiations (RFR), such as those emitted by wireless communication devices. Since the introduction of mobile phones many studies have been conducted regarding alleged health effects but there is still some uncertainty and no definitive conclusions have been reached so far. Although thermal effects are well understood they are not of great concern as they are unlikely to result from the typical low-level RFR exposures. Concern rests essentially with the possibility that RFR-exposure may induce non-thermal and/or long-term health effects such as an increased cancer risk. Consequently, possible genetic effects have often been studied but with mixed results. In this paper we review the data on alleged RFR-induced genetic effects from in vitro and in vivo investigations as well as from human cytogenetic biomonitoring surveys. Attention is also paid to combined exposures of RFR with chemical or physical agents. Again, however, no entirely consistent picture emerges. Many of the positive studies may well be due to thermal exposures, but a few studies suggest that biological effects can be seen at low levels of exposure. Overall, however, the evidence for low-level genotoxic effects is very weak.

Fractionation effects and repair kinetics in rat kidney.

Rat kidneys were unilaterally irradiated with up to 40 fractions of X rays. Fractionation regimens were given either with long intervals of 6-24 hr between fractions, resulting in complete recovery from sublethal damage, or with 1-hr intervals, resulting in largely incomplete repair. The non-irradiated kidney was surgically removed 4 weeks after the last fraction. The development of radiation-induced kidney damage was monitored by regular assessment of three different parameters indicative of kidney function: serum urea, the total volume of urine excreted in 24 hr and urine osmolality. At the end of the observation period, 18 months after treatment, the kidney was removed. The hydroxyproline content was determined and a histopathological analysis was performed. Since the 20 and 40-fraction data indicated a higher effectiveness of these regimens than would be expected on the basis of the LQ model, the data were divided in two subsets, 2-10 fractions (high doses per fraction) and 10-40 fractions (low doses per fraction), and analyzed separately. The time course of alpha/beta and T1/2 values was determined for each functional parameter separately, and for the data from the three parameters combined. A complex pattern was found, with the values for alpha/beta as well as T1/2 differing between the two data subsets between about 20 and 40 weeks after treatment. For the lower doses per fraction the alpha/beta values were generally higher and the repair half-times longer. After 40 weeks no significant differences were observed between the two data subsets. If the differences found earlier are ignored, overall alpha/beta and T1/2 values can be calculated. For early endpoints the alpha/beta was 1.69 (1.45, 1.90) Gy (95% confidence limits in parentheses), for late endpoints it was 1.77 (1.56, 2.00) Gy. The corresponding T1/2 values were 1.57 (1.44, 1.73) hr for early endpoints, and 2.10 (1.90, 2.34) hr for late endpoints. Hence, the alpha/beta values do not alter in time, but the T1/2 value for late damage might be higher than that for early damage.

Effects of multifraction irradiation on the rat kidney.

Wag/Rij female rats were irradiated to the left kidney with single doses or 2, 4, 10, 20, or 40 equal dose fractions. The right kidney was removed 4 weeks after the last fraction. The kidney function was determined using three different parameters. The serum urea content indicated glomerular function. Urine osmolality and the total volume of urine excreted in 24 hr indicated tubular function. The onset as well as the rate of expression of radiation-induced kidney damage was dose-dependent. The kidney function decreased continuously. Differences in expression of damage between glomerular and tubular parameters were not observed. All parameters indicated marked sparing of the kidney by fractionation. In general, the data could be fitted to the linear-quadratic model, if the single dose data were not included in the analyses. However, the fit greatly improved when data obtained with high and low doses per fraction were analyzed separately. The Direct Analysis method was used to determine the alpha/beta ratios. No significant differences were observed between the alpha/beta ratios calculated for the different parameters. The ratios also did not change with increasing time after treatment. The alpha/beta for high doses per fraction was between 0.6 and 2.7 Gy, and that for low doses per fraction, with fractional doses in the clinical range, was between 0.5 and 3.8 Gy. The alpha/beta values for low doses per fraction were generally lower than those for high doses per fraction. These observations indicate a strong dependence of radiation-induced damage in the rat kidney on the size of the dose per fraction.

Acute reactions in rat feet exposed to multiple fractions of X-rays per day.

Acute reactions of rat foot skin to regimens of X-irradiation involving single and multiple fractions per day were determined. The influence of an interval halfway through the treatment course was investigated for a multiple fractions per day regimen. The regimen was one in which 4 fractions of 2 Gy were given each day. Two courses of up to the tolerance dose separated by an interval of several weeks can be given without causing excessive skin damage. Repopulation during the interval was found to be responsible for the increase in tolerance. Repopulation started at about 2 weeks after the beginning of treatment. Using Barendsen's isoeffect model [4], doses which when given daily would result in the same biological effect as 4 X 2 Gy given daily, were calculated. Rat feet were irradiated with these doses. It was concluded that the isoeffect model provides good predictions of the tolerance doses in new irradiation regimens for the acute reaction of the rat foot skin.

Combined interstitial 125I and external X-irradiation on a rat rhabdomyosarcoma.

The effect of graded external radiation doses and a dose administered by an implanted 125I seed on growth delay and cure rate of a rat rhabdomyosarcoma was investigated. One 125I seed (0.40 or 0.50 mCi) was implanted per tumour when the tumours had grown to a predetermined size. The irradiation by the 125I seed did not cause significant tumour growth delay. When implantation of an 125I seed was combined with graded external radiation doses, the growth delay observed after these combined regimens was significantly greater than that observed after treatment with external doses alone. The same was observed for tumour cure rates. The effectiveness of the 125I seed could be assessed as being equivalent to a single dose of external radiation of about 6-20 Gy, depending on the external dose applied. The application of 125I seeds combined with external irradiation to obtain local control might be recommended in those cases where the total dose of external radiation alone is limited by normal tissue reactions.

The influence of platinum drugs on the radiation response of rat kidneys.

The influence of a single bolus injection of platinum drugs on the radiation sensitivity of the kidneys was investigated in WAG/Rij rats. Drugs employed were cis-diammine-dichloroplatinum(II) (cisplatin, CDDP), cis-diammine-1,1-cyclobutanedicarboxylate platinum(II) (carboplatin, CBDCA) and cis-dichloro,trans- dihydroxy-bis-isopropylamine platinum(IV) (iproplatin, CHIP). Both kidneys were irradiated with a range of single X-ray doses while drugs were administered at 1 day or 1 week before irradiation. Maximum tolerated drug doses (defined as the LD1, the dose resulting in a mortality of 1%) were given. Damage inflicted upon the kidneys was monitored by determination of several parameters indicative of kidney function. Isoeffective radiation doses were calculated from these data for each treatment group at 4-8-week intervals up to 80 weeks following treatment. At each assay time, dose modifying factors (DMF) were calculated for each drug/radiation combination. The mean DMFs were highest for CDDP: approximately 1.6. Those for CBDCA and CHIP were lower: approximately 1.1 and 1.2, respectively. The CHIP DMFs were significantly different from unity. When the radiation was given in 4 or 8 daily fractions (4 fractions/week) the DMFs for CDDP were identical to those obtained with single doses. For CBDCA and CHIP, however, the DMFs after fractionated treatments were not significantly different from unity. Analysis in terms of the linear-quadratic (LQ) model indicated that not one of the three drugs had an effect on the alpha/beta ratio, and hence on the fractionation sensitivity of the rat kidney. Consequently, if these data are extrapolated to the clinical setting, the administration of these drugs at the maximum tolerated dose preceding a fractionated radiation treatment should not be expected to result in extra, unexpected, radiation toxicity of the kidney.

Late functional, biochemical and histological changes in the rat lung after fractionated irradiation to the whole thorax.

The influence of fractionation of the radiation dose in up to 16 fractions on late occurring effects in rat lungs was studied. Measurement of the breathing frequency (BF) was used to monitor changes in lung function during the 76-wk follow-up period. At the end of this period the lungs were examined histologically and the hydroxyproline (HP) content of the lung tissue was determined as a biochemical indicator of the amount of fibrosis. Dose-response curves for BF could only be made at 76 wk post-irradiation. No dose-response curves could be constructed for HP/mg dry tissue; dose-related increases observed in HP/total left lung were entirely attributable to increases in lung dry weight. Histopathology data indicated a dose-related increase in two histological alterations, septal fibrosis and granulomatous pneumonia. The alpha/beta ratios were calculated for each of the endpoints using both the Fe-plot and Direct Analysis method. The calculated values for alpha/beta were in the range of 1.1 to 3.6 Gy, corresponding well to published data obtained in mouse lungs. The number of tissue rescuing units (TRU's) was also calculated. It was smaller for BF than for the histopathology endpoints, suggesting different target structures. However, the difference was not significant due to large confidence intervals.

Early and late effects of fractionated irradiation and the kinetics of repair in rat lung.

The thorax of WAG/Rij rats was irradiated with fractionated doses of X rays. Irradiation schedules were designed either to allow virtually complete repair of sublethal damage between subsequent fractions by fractionating at 6-h intervals, or to result in incomplete repair by allowing only 1-h intervals between subsequent fractions. Combination of the data from both experimental series permitted the calculation of alpha/beta ratios and values for the repair halftime T1/2. The animals were monitored by assessment of the breathing frequency and by recording deaths. At the end of the experiments, 18 months after treatment, the hydroxyproline content of the lung tissue was determined as a biochemical indicator of radiation-induced fibrosis, and an histopathological analysis was performed. Early endpoints, indicative of radiation-induced pneumonitis, resulted in an alpha/beta ratio of 3.5 Gy and a T1/2 value of 0.95 h. Late endpoints were presumed to be indicative of radiation-induced fibrosis. Based on the combined analysis of data from three different late endpoints, the mean alpha/beta ratio was 2.3 Gy, and the T1/2 value was 1.13 h. The difference in alpha/beta ratio and T1/2 value between early and late endpoints was not significant, since the 95% confidence limits were overlapping. For each individual early or late endpoint as well as for the two early or the three late endpoints combined, there was a trend for lower alpha/beta ratios and higher T1/2 values associated with low doses per fraction. However, widely overlapping confidence limits indicated that again the differences were not significant.

Analysis of cell survival after multiple fractions per day and low-dose-rate irradiation of two in vitro cultured rat tumor cell lines.

Two rat tumor cell lines which differ significantly in radiosensitivity, a rhabdomyosarcoma (R-1) and a ureter carcinoma (RUC-2), were treated with multiple fractions per day and low-dose-rate gamma radiation. The purpose of these experiments was to investigate (i) the influence of fraction size and interfraction interval on repair of sublethal damage (SD) and (ii) whether low-dose-rate irradiation can be simulated by giving multiple fractions per day which might be applied in clinical treatments. In both cell lines, multiple doses were given at 1- to 4-hr intervals. SD repair was at a maximum in 2 hr but did not reach the theoretically expected level. For both cell lines, survival at higher total doses was different from that theoretically expected if repair of SD was assumed to be completed and at the maximum level. To account for the observation that less than complete repair of SD occurred, theoretical survival curves were calculated with the assumption of a constant but less than 100% level of SD repair. Experimental data correlated well with these calculated curves. There were only very small differences in survival after the different multiple fractions per day regimens. Survival after irradiation at a dose rate of 1.00 Gy/hr was found to be similar to that after multiple fractions per day.

Repair rate in mouse lung after clinically relevant radiation doses per fraction.

Data published previously have shown that repair of sublethal damage in mouse lung proceeds with two significantly different repair half-times of 0.4 h and 4.0 h and that the fast component has approximately four times more weight than the slow component. None of these data, however, were obtained after small dose fractions similar to those used in clinical radiotherapy. The purpose of the experiments presented here was to determine the half-time of the fast component only of repair in mouse lung after doses per fraction of 2.0 Gy. We irradiated the whole thoraces of mice with six equal doses of 2.09 Gy given at intervals ranging from 0 to 45 min. The dose was topped up 24 h later by a range of single doses designed to bring the response, i.e. breathing rate and death from pneumonitis, into the observable range. Data on breathing rate were converted into quantal response data. All data were analyzed by the linear-quadratic model that contains two rates of repair (H.D. Thames et al., Radiother, Oncol. 15, 49-53, 1989). The data showed that the repair rate is very rapid, giving a t1/2 ranging from 0.25 to 0.75 h for breathing rate and mortality, in agreement with our data published previously for higher dose fractions. There were no differences between the t1/2's obtained from the two assays of damage. These data indicate that the half-time of the fast component of repair in mouse lung is approximately 0.4 h after clinically relevant dose fractions.

Recovery from radiation damage in mouse lung: interpretation in terms of two rates of repair.

A reanalysis was performed of the extensive data set obtained with fractionated irradiations of mouse lung reported by Travis et al. (Int. J. Radiat. Biol. 52, 903-919, 1987). The possibility was investigated that the poor fit of these data to the linear-quadratic model might have been the result of the presence of two rates of repair of sublethal damage instead of one. Therefore, the incomplete-repair linear-quadratic model was adapted to incorporate two independent rates of repair and the data were analyzed using this two-component incomplete-repair model. The results which are subjected to certain qualifications with respect to the assessment of the validity of the confidence limits indicated the presence of two significantly different repair rates, corresponding to a fast-repair half-time (t1/2) of 0.40 h (0.28, 0.53) and a slow t1/2 of 4.01 h (1.55, 6.57). A weight factor determined simultaneously indicated that the fast component has approximately four times more weight than the slow component. The alpha/beta value calculated for the entire data set using the same model was 3.8 Gy (3.0, 4.6), which is not significantly different from the alpha/beta of 3.6 Gy (2.8, 4.5) calculated for the 8- and 12-h data only, using the complete-repair linear-quadratic model. An experiment specifically designed to test the significance of the fast-repair component was performed in which mouse lungs were irradiated with two equal dose fractions, separated by intervals ranging from 10 min to 6 h. Data obtained from this experiment allowed only one repair rate to be determined, corresponding to a t1/2 of only 0.4 h. This finding confirms the presence of a very fast repair rate in mouse lung.

Xenotransplantation: perspectives from The Netherlands.

Recently, the Health Council of the Netherlands published a report on xenotransplantation. The technique is considered ethically acceptable, both from a human and from an animal point of view. Clinical application should not yet be permitted, however, because of continuing rejection problems and growing concerns on safety aspects associated with possible transfer of pathogens from the xenotransplant to the host. More research is necessary. Mainly because of the risk for infection, nonhuman primates are not considered acceptable as source animals; the genetically engineered pig is at present the source animal of choice. Legally, import in the Netherlands of (organs from) source animals from within Europe is difficult to prevent. Therefore experiments with such material are not subjected to licensing by the government, which is the case for experiments involving genetic engineering with animals in the Netherlands, but only by local review boards. This situation is considered unwanted and adjustment of the pertinent law is suggested. Also, both genetically engineered animals and patients carrying organs from such animals fall under the European Genetically Modified Organisms regulations. These regulations are not designed for this case and conflicts may arise. Agreements on how to handle this situation have to be made at the European level. It is proposed to draft a national law on "biologicals," medical products consisting at least partly of living materials. Pending such regulations, it is suggested, also on a European level, that xenotransplants be considered as medicines, which would allow adequate control of safety aspects.

Time- and sequence-dependent responses to cisplatin and radiation in the rat kidney.

The influence of time interval and sequence between administration of cisplatin and a radiation dose was studied in the rat kidney. A dose of 10.5 Gy X-rays was given to both kidneys, preceded or followed by a single dose of cisplatin. Two separate experiments were performed. In the first experiment 6.0 mg/kg cisplatin was given, in the second experiment the drug dose was 5.5 mg/kg. A range of time intervals was introduced between administration of drug and radiation, from 7 to 1 days, 12 to 1 h, and 30 to 0 min. Control animals received either modality alone, or were left untreated. Cisplatin alone caused tubular function to decrease very quickly and to remain permanently altered. Changes in glomerular function were only detected after 30 weeks following the higher drug dose. X-rays alone caused measurable alterations in both glomerular and tubular function after 16 weeks. In the combined treatment the influence of time and sequence was significant. If cisplatin was given at 7 to 1 days before X-rays the effect of time was minimal. Administration of cisplatin 12 h to 15 min before irradiation resulted in an increase of radiation damage with decreasing time interval. Total damage sharply decreased when both modalities were given at the same time, and decreased further with increasing time between irradiation and drug administration. It is suggested that in the tubular cells free cisplatin or one of its hydrolysis products may interact with radiation-induced damage, e.g. by interference with repair of sublethal or potentially lethal damage.

The influence of cisplatin and unilateral nephrectomy on the response of the rat kidney to irradiation.

Cisplatin was administered as a single i.p. dose of 5 mg/kg to WAG/Rij rats at intervals of 7 days or 0.5 h before, or 7 days after graded X-ray doses to the left kidney. The right kidney was surgically removed 4 weeks after irradiation. Renal function was determined by measuring total urine volume excreted in 24 h, urine osmolality and serum urea. The severity of alterations in the various anatomic compartments of the kidney induced by the various treatments were graded histologically. The administration of 2 or 5 mg/kg cisplatin alone did not alter any of the kidney function parameters. Isoeffective radiation doses calculated for each of the functional parameters continuously decreased with increasing time after treatment. Differences between the isoeffective doses for the three combined treatments and for treatment with irradiation alone were only observed for urine osmolality and urine volume which primarily are tubular-related functional parameters. The histopathological grading studies also indicated that enhancement by cisplatin of radiation-induced damage was almost entirely confined to the tubules. The results of this study indicate that the sequence and length of time between treatments is an important variable in the development of cisplatin plus radiation-induced renal injury, but that none of the tested combinations showed a more than additive toxicity.

Responses of cultured mammalian cells to prolonged low dose rate gamma irradiation.

Experiments were performed to verify the finding of others that prolonged irradiation caused sudden death of cells. Continuous irradiation (dose rate 0.09 Gy/h) was applied to the R-1, RUC-2, V79 and T-1g cells for up to 140 days. End points were: population growth rate, plating efficiency and radiosensitivity. The population doubling times were increased and plating efficiencies decreased; cell survival after single doses at acute dose rate were lower than that of control cells. Part of the cell parameters returned to control values within three weeks after prolonged irradiation had been terminated. Sudden death after prolonged exposure was not observed.

Interaction of cisplatin and x-rays in rat kidney.

Cisplatin was administered as a single iv dose of 5 mg/kg in WAG/Rij female rats at intervals of 7 days or 30 minutes before or 7 days after graded irradiation of the left kidney. The unirradiated right kidney was removed 4 weeks after the x-ray treatment. Kidney function was determined by measuring urine osmolality and plasma urea. The kidney function parameters did not change measurably in animals treated with cisplatin alone. Only differences in urine osmolality were observed between the groups that received combined treatment or irradiation only. Long-term renal fibrosis was assessed by measuring the hydroxyproline content. Significant increases in renal hydroxyproline content were observed in animals receiving treatment with cisplatin either 7 days before or 7 days after irradiation, compared with animals receiving irradiation alone.

Lung tumour growth delay and normal tissue toxicity induced by three cytotoxic platinum drugs.

Single doses of the drug cisplatin and its analogues carboplatin and iproplatin were administered to tumour-bearing rats. The tumours used were two bronchial squamous cell carcinomas, that are part of a panel of experimental lung tumours developed at this institute. Cisplatin resulted in severe nephrotoxicity. Carboplatin and iproplatin were less nephrotoxic, but resulted in acute gastrointestinal and (probably) hematological toxicity. Carboplatin also caused late occurring liver damage. The responses of the tumours were compared at the level of maximum tolerated drug doses for early toxicity. The level of response was different for the two tumours. One was more sensitive to the drugs than the other. The effects of cisplatin and carboplatin were approximately similar. Iproplatin was less effective. Because cisplatin caused more severe late toxicity, it is concluded that carboplatin has the best therapeutic index for these two lung tumours.