Requirements regarding dose rate and exposure time for killing of tumour cells in beta particle radionuclide therapy Purpose The purpose of this study was to identify combinations of dose rate and exposure time that have the potential to provide curative treatment with targeted radionuclide therapy applying low dose rate beta irradiation . Introduction Many types of tumour overexpress cell surface-associated antigens or receptors suitable as targets for radionuclide therapy , and many types of targeting agent have been suggested or are already being applied for such therapy . This therapy is currently employed for lymphomas -LSB- 1 , 2 -RSB- using radiolabelled antibodies and also for neuroendocrine -LSB- 3 -- 5 -RSB- and paediatric tumours -LSB- 6 , 7 -RSB- using radiolabelled somatostatin analogues and meta-iodobenzylguanidine -LRB- mIBG -RRB- , respectively . In the majority of these cases , beta emitters such as 90Y , 131I and 177Lu have been applied . The results have , so far , essentially shown palliative effects -LSB- 1 , 8 -- 12 -RSB- , and there is hope that combinations of beta particle emitters , e.g. 90Y and 177Lu , will improve the therapy results -LSB- 13 -RSB- . The cell-killing capacity of low LET radiation , i.e. photons and electrons , is well known when applying high dose rates , typically 0.5 -- 2.0 Gy/min , as in external radiotherapy -LSB- 14 , 15 -RSB- . However , the extensive experimental and clinical knowledge on the effects of external radiotherapy can be deployed to only a limited extent in understanding the effects of radionuclide therapy . A major difference is that the dose rate in radionuclide therapy is at least two orders of magnitude lower than in external radiotherapy -LSB- 10 , 16 -- 19 -RSB- . The lower dose rate allows for DNA repair and repopulation during the radiation exposure , which is not the case during high dose rate exposures . Basic radiobiological studies have shown that low dose rates , in the range of 0.1 -- 1.0 Gy/h , give a much lower biological effect -LRB- per dose unit -RRB- than high dose rates in the range 0.5 -- 2.0 Gy/min -LSB- 15 , 17 , 20 , 21 -RSB- . It is also known that an inverse dose rate effect exists in that dose rates of 0.2 -- 0.4 Gy/h can give more cell kill than dose rates in the range 0.7 -- 1.0 Gy/h -LSB- 15 , 22 -RSB- . Only crude estimates can be made from previous experiments to elucidate which combinations of low dose rate and exposure time can cure a metastasis containing , for example , 105 cells . Cell survival has most often been analysed after a cell cloning 1 -- 2 weeks after the radiation exposure . For example , a total dose of about 30 -- 50 Gy , given with 0.1 -- 1.0 Gy/h , seems necessary to decrease the single cell survival probability to 10 − 5 -LSB- 23 , 24 -RSB- , and thereby give a reasonable chance of killing 105 tumour cells . Furthermore , targeted radionuclide therapy is complicated , since it is not enough only to consider the macroscopic dose concept ; different cellular and intracellular distributions of radionuclides can give different biological effects although the macroscopic dose is the same -LSB- 25 , 26 -RSB- . One way to obtain solid information on which combinations of low dose rate and exposure time can give curative treatments with beta particles -LRB- which also is low-LET radiation -RRB- is , of course , through experiments and clinical trials . In this study we used an experimental model with the criterion that the low dose rate beta radiation must kill all 105 tumour cells in a culture dish in order to simulate a successful treatment . The follow-up period was 3 months . The choice of 105 tumour cells is somewhat arbitrary and is based on two arguments . The first is that this number represents a small tumour cell cluster that normally can not be identified by routine diagnostic procedures such as computed tomography or magnetic resonance imaging -LRB- unless the tumour cells cause macroscopic changes in the surrounding normal tissues -RRB- . Furthermore , this number of tumour cells in most cases does not cause symptoms in the patient . Thus , a cluster of 105 tumour cells in a patient can be considered an `` occult '' or `` subclinical '' tumour or metastasis . The second argument is more practical , since the presence of 105 tumour cells in a normal cell culture dish or flask allows enough space for exponential growth and , at the same time , frequent cell -- cell contacts . Our ambition was not to simulate the dose rate variations in time and space that occur in radionuclide therapy . In the clinical setting , the dose rate varies with time , not only as a consequence of the physical half-life of the radionuclides , but also due to time-dependent changes in their spatial distribution -LSB- 16 , 17 , 24 , 26 , 27 -RSB- . Factors of importance are ongoing vascularisation processes , variations in vessel wall leakage and changes in blood flow . There are probably also various diffusion and convection conditions in different areas of tumours , resulting in variable penetration properties of the radiolabelled targeting agents . In addition , there might be variations in the expression of target structures on the tumour cells . All these time-dependent factors make it difficult to establish basic and reproducible dose rate -- response relations in vivo . Our experimental model was designed to give reproducible and controllable irradiation conditions , and we applied a model with a rather long physical half-life -LRB- 32P sources with T1/2 = 14.3 days -RRB- , giving only a slow decrease in dose rate during the exposures . Relevant dose rates were selected through the amount of radionuclide placed in the irradiation chambers . The exposure times were selected to correspond to the effective half-lives of the radionuclides delivered by targeting agents of different types . In targeted radionuclide therapy it is , of course , also necessary to consider unwanted side-effects on normal tissues . However , analyses of normal tissue effects were beyond the scope of this study . Hyperradiosensitivity -LSB- 28 , 29 -RSB- at low doses , bystander effects -LSB- 30 -- 32 -RSB- and low dose rate-induced apoptosis -LSB- 33 , 34 -RSB- are all extensively studied processes . Our model allows these processes to work together , but we did not try to study them separately . The overall goal of the study was to find `` dose rate -- exposure time '' relations that could kill all of the exposed 105 tumour cells , so that no remaining cells would be observed after 3 months . Materials and methods Irradiation chambers The irradiation chambers have been described previously -LSB- 35 -RSB- , so only a short description is given here . Three identical chambers were used and each was filled with 400 ml distilled water containing 0.74 -- 2.22 GBq 32P . The water was boiled and degassed shortly before adding 32P and filling of the chambers , in order to avoid air bubbles . The beta emitter 32P -LRB- orthophosphate -RRB- -LRB- T1/2 = 14.3 days -RRB- was obtained from Amersham Pharmacia Biotech -LRB- Amersham , UK -RRB- . The upper area of the chambers , where the cells were exposed to beta particles from 32P , was covered with a 0.5-mm thin transparent polycarbonate foil . The radiation protection walls surrounding the side and the bottom of the chambers , as well as the lid above the cell cultures , consisted of 15-mm transparent polycarbonate -LRB- Macrolon -RRB- . 32P could not reach and be incorporated in the cells . The irradiated cells were grown in 3-cm-diameter culture dishes with a plastic bottom thickness of 1 mm . The culture dishes were placed directly on the thin foils above the 32P source . The chambers were kept in cell culture incubators at 37 °C -LRB- Kebo Assab T304GF , Stockholm , Sweden -RRB- and supplied with 5 % carbon dioxide . The lid above the cultures had side openings , allowing efficient passage of the incubator atmosphere . Dosimetry The dosimetry has also been described previously -LSB- 35 -RSB- . The dose rate for each chamber was controlled by measurements with a thin-walled parallel ion chamber -LSB- 36 -RSB- . The ion chamber was calibrated using an external 60Co source with the front wall of the ion chamber at the dose maximum depth , where the dose rate was 0.30 Gy/min . The 60Co source was calibrated according to national standard procedures accepted for radiotherapy purposes . The thin-walled parallel ion chamber accurately measured the dose independent of dose rate down to at least 0.01 Gy/h , if corrections were made for `` leakage current '' in the instrumentation . The measurements of dose rates were performed with the thin-walled ion chamber placed in cell culture dishes standing on the 32P radiation chambers . This procedure allowed mimicking of the dose rate in the cell environment . All three chambers were measured repeatedly . Measurements were also made with a surface hand detector -LRB- RNI 10/R Intensimeter , Nuklex , Uppsala , Sweden -RRB- , and a calibration curve was constructed to facilitate repeated dose rate determinations during the cell culture periods . Tumour cells The cells used were HT-29 colorectal adenocarcinoma , A-431 cervical squamous carcinoma , SKBR-3 breast cancer , all from the American Type Culture Collection -LRB- ATCC -RRB- , and the two gliomas U-118MG and U-373MG from the Department of Pathology , Uppsala University , Uppsala , Sweden . They were grown in Ham 's F-10 medium supplemented with 10 % fetal bovine serum , 2 mmol/l L-glutamine , 100 μg/ml streptomycin and 100 U/ml penicillin , all components from Sigma AB -LRB- Stockholm , Sweden -RRB- . The cells were normally grown in an incubator of type Galaxy S -LRB- LabRum Klimat AB , Stockholm , Sweden -RRB- , and during the irradiations they were grown in a similar incubator -LRB- Kebo Assab T304GF , Stockholm , Sweden -RRB- . Both incubators were run at 37 °C and supplied with 5 % carbon dioxide . The cells in the study were selected for the following reasons . SKBR-3 cells are often studied since they express large amounts of HER2 receptors and are therefore applied in experiments on HER2-directed radionuclide targeting , using antibodies or affibody molecules -LSB- 37 , 38 -RSB- . A-431 cells express both large amounts of EGFR and also rather large amounts of HER2 and are therefore often studied for radionuclide targeting with both EGF ligands -LSB- 39 -RSB- and anti-HER2 antibodies -LSB- 40 -RSB- . Both cell types are planned for experimental therapy using beta particle-mediated radionuclide therapy , especially 177Lu . The two glioma cell lines also express EGFR -LSB- 41 -RSB- to some degree but were , together with the HT-29 cells , selected because they have recently been studied at our laboratory with regard to short-term effects after low dose rate irradiations -LSB- 35 -RSB- . It was found that U-373MG cells showed radiation-induced apoptosis , while U-118MG cells did not . The HT-29 cells were intermediate in this respect -LSB- 35 -RSB- . Furthermore , U-373MG cells have previously been reported not to show hyperradiosensitivity at low doses , while both U-118MG and HT-29 cells do -LSB- 28 -RSB- . Choice of dose rate and exposure time In the previous study , with the same 32P chambers , we analysed effects on cells after exposure to initial dose rates of only 0.05 -- 0.09 Gy/h for 7 days -LSB- 35 -RSB- . The cells in that study were analysed for effects on cell number , apoptosis and cell cycle block at day 7 of continuous exposure . However , we also allowed cells to continue to grow for longer times in parallel dishes and found that all cultures recovered . Thus , we decided to apply initial dose rates from 0.1 Gy/h up to 0.8 Gy/h in the present study to have a reasonable chance of `` simulating '' curative treatment . We could not apply higher dose rates because of regulatory rules limiting the amount of 32P that could be handled in the cell culture laboratory . Furthermore , this dose rate range is the same as that applied in published studies on low dose rate effects -LSB- 21 -- 23 -RSB- , hyperradiosensitivity and low dose rate -LSB- 29 -RSB- , and radionuclide treatment of gliomas in vitro -LSB- 42 -RSB- and in vivo -LSB- 43 -RSB- , and actually covers the range up to the highest dose achievable in targeted radionuclide tumour therapy -LSB- 10 , 16 , 18 , 27 , 44 -RSB- . Four cell dishes were placed in each irradiation chamber when the dose rate was as expected and the cells were then kept there for 24 , 72 or 168 h -LRB- 1 , 3 or 7 days -RRB- . The radiation exposure time was chosen not to be longer than a week , since it is known from numerous articles in the field of radionuclide therapy that the tumour cell retention of radioactivity is generally in the range of some days up to about 1 week -LRB- in some cases it is only a few hours -RRB- . Furthermore , it is well known that the biological half-life of tumour targeting agents -LRB- ligands , antibodies and antibody fragments -RRB- in the systemic circulation is often shorter than a week -LRB- in the case of small ligands , it is only a few hours -RRB- . Thus , the longest exposure time considered to be realistic was 1 week . Cell counting The medium was removed from the cell dishes and the cells were quickly washed with 0.5 ml trypsin-EDTA -LRB- 0.25 % trypsin/0 .02 % EDTA solution in PBS , VWR , Stockholm , Sweden -RRB- and then incubated with 0.5 ml trypsin-EDTA -LRB- 37 °C , 5 % CO2 -RRB- until the cells detached . Next , 1.5 ml medium was added to each dish and the cells resuspended to a single cell solution . For cell counting , 19.5 ml PBS -LRB- pH 7.4 -RRB- was added to 0.5 ml cell suspension and an electronic cell counter was used -LRB- Coulter Z2 , 7 -- 20 μm , Beckman Coulter , Stockholm , Sweden -RRB- . Cell culture conditions during and after irradiations Cells were seeded sparsely in culture dishes -LRB- diameter 3.5 cm , surface 9.6 cm2 , Nunc , Roskilde , Denmark -RRB- a few days before the start of the radiation exposure . They were seeded so that each culture dish contained about 105 cells at the start of the exposure . The first cell count was made at the start of each experiment , on representative culture dishes . If the cell number was about 105 per dish , four other , parallel culture dishes were placed in each of the three irradiation chambers . The irradiated cells were grown in the chambers with unirradiated control dishes placed on a near radiation-shielded shelf in the same incubator . The cells did not reach confluence during the irradiation period . After the radiation exposure , the cells were moved from the irradiation chamber incubator to another incubator and the cell growth was followed for several months . At the first subcultivation after the radiation exposure , the cells were transferred from culture dishes to culture flasks -LRB- 25 cm2 , Nunc , Roskilde , Denmark -RRB- , and these flasks were then used throughout the growth period . The medium in all culture dishes and flasks was replaced three times a week . Cell counting was in most cases performed once a week , followed by reseeding of 105 cells in each new flask . Growth curves The growth curves were constructed as if all cells had been saved at each subcultivation . By calculating how many cells would have been obtained if all cells had been saved , such high cell numbers as 1015 -LRB- Fig. 1a -RRB- and even up to 1025 -LRB- Fig. 1b -RRB- were obtained . In reality , repeated dilutions were made to keep the number of cells in each culture flask in the range of 105 -- 106 , thus allowing for exponential growth . Four parallel flasks were kept for each experiment . In order to analyse the significance of the differences between the groups , t tests were performed . Cell kill versus regrowth The criterion for killing of a cell culture with 105 cells was that no living cells could be seen in the phase contrast microscope and that no regrowth was observed after at least 3 months of follow-up , also applying electronic cell counting . Growth delay Growth delay was also analysed after 1 , 3 or 7 days of continuous low dose rate exposure . The growth curves of the irradiated samples in some cases did not have the same slope as the control curves , so it was not possible to wait and measure growth delay when the recovered cells and the control cells had a similar growth rate . Instead , growth delay was defined as the time it took for the irradiated cells to reach the cell number 1010 in relation to the time it took for the control cultures to reach this number . Literature survey The Medline-based PubMed database was used to survey effects of targeted radionuclide therapy and of low dose rate therapy . Results Growth curves Figure 1 shows examples of growth curves for control cells and cells exposed to continuous low dose rate beta irradiation . In Fig. 1a the initial dose rate was 0.415 Gy/h and the exposure time 72 h . After 72 h the dose rate had decreased to 0.359 Gy/h , and the exposure gave a total dose of about 27.8 Gy . The irradiated HT-29 cells recovered after irradiation and resumed , after about 50 days , a similar growth rate as the controls . In contrast , the U-373MG cells completely ceased to grow after the same radiation exposure . After more than 30 days , only a few giant cells could be identified in the microscope . They were too few to be counted with the electronic cell counter . However , there was a possibility that at least a few of these cells could grow later , so they were observed -LRB- with normal medium changes -RRB- for up to 3 months . Nevertheless , no recovery could be observed ; instead , the cells disappeared . It was then concluded that the irradiation procedure had killed all U-373MG cells . Thus , the HT-29 cultures survived the treatment , while the U-373MG cells died . Fig. 1Examples of cell growth as a function of time after exposure to low dose rate irradiation . a HT-29 and U-373MG cells were exposed to 0.415 Gy/h as the initial dose rate and then continuously exposed for 72 h. b A-431 , U-118MG and SKBR-3 cells were exposed to 0.806 Gy/h as the initial dose rate and then continuously exposed for 24 h. Mean values and maximal variations from four parallel samples are given Figure 1b shows examples of growth curves for an initial dose rate of 0.806 Gy/h and an exposure time of 24 h . After 24 h the dose rate had decreased to 0.768 Gy/h and the exposure gave a total dose of about 18.9 Gy . The irradiated A431 cells continued to grow , but at a slower rate than the controls . The growth of irradiated U-118MG cells was arrested for up to about 40 days after the treatment . During those 40 days , resting cells were observed in the culture flasks , but there was no sign of growth . After 40 days a few mitotic cells were observed in the phase contrast microscope , and the number of cells was thereafter measured with the electronic cell counter . A clear regrowth was seen after 60 days . However , their growth rate was slower than that of the corresponding controls . The SKBR-3 cells looked severely damaged -LRB- fragmented , decreasing in number and forming giant cells -RRB- some days after the exposure and were too few to be counted with the electronic cell counter . Within 1 month , no surviving cells could be observed in the culture flasks . In this case , too , we continued to control the culture flasks for 3 months -LRB- with normal medium changes -RRB- and found that there were no remaining cells in these cultures . Thus , A-431 and U-118MG cells survived , while the SKBR-3 cells died . The conclusions from the examples in Fig. 1a and b are that four different responses were observed : The HT-29 cells recovered to the control growth rate after a growth delay.The U-118MG cells recovered after a growth delay but continued to grow at a slower rate than the controls.The A-431 cells continued to grow without delay but at a slower rate than the controlThe U-373MG and SKBR-3 cells died . Cell kill and regrowth versus dose rate and exposure time Figure 2 summarises cell-killing and regrowth results from all performed low dose rate experiments . All five analysed cell types were irradiated with different initial low dose rates -LRB- 0.1 -- 0.8 Gy/h -RRB- and were continuously exposed for 1 , 3 or 7 days . Fig. 2Summary of all low dose rate experiments carried out for a U-118MG , b U-373MG , c HT-29 , d A-431 cells and e SKBR-3 cells . The cells were irradiated with different initial dose rates and were then exposed to the radiation for 1 , 3 or 7 days . The figures show at which combinations of dose rate and exposure time all cells were killed -LRB- area with no survivors -RRB- , and at which at least some cells survived and displayed regrowth -LRB- the regrowth area -RRB- . The separation between the two areas is indicated by bold solid lines . The total delivered radiation dose -LRB- Gy -RRB- is given in parentheses near each point . The 20-Gy isodose curve is indicated by a dashed line Figure 2 parts a -- e show a similar and general pattern . When applying 7 days of continuous irradiation , low initial dose rates , about 0.2 -- 0.3 Gy/h , were enough to kill all cells . When cells were exposed for only 3 days , a dose rate in the order of 0.4 -- 0.6 Gy/h was necessary to kill all cells . Only the SKBR-3 cells were killed after 24-h exposure to about 0.8 Gy/h . As mentioned in Materials and methods , higher dose rates could not be used in these experiments , so we do not know how high the dose rates would have to be during 24-h exposure in order to kill the other cell types . It was found that the regrowth pattern was the same in all four flasks . However , even if the results looked rather similar and independent of cell type , there were some cell type-dependent differences . The U-118MG cells -LRB- Fig. 2a -RRB- required , during 7 days ' exposure , at least about 0.3 Gy/h as the initial dose rate to be completely growth inactivated . The U-373MG cells -LRB- Fig. 2b -RRB- were completely growth inactivated after an initial dose rate of only about 0.15 Gy/h and 7 days ' exposure . Thus , the U-373MG cells were , in these cases , easier to kill than the U-118MG cells . Considering all cells , it seemed that the U-118MG cells were most resistant and U-373MG and SKBR-3 most sensitive , while A-431 and HT-29 were intermediate . Cell kill and regrowth versus total dose The 20-Gy isodose curve is drawn with a dashed line in Fig. 2a -- e. For U-373MG and SKBR-3 cells , at least a total dose of 20 Gy was necessary to prevent regrowth . For HT-29 and A-431 cells an even higher total dose seemed necessary , and for the resistant U-118MG cells it seemed that total doses of at least 40 Gy were necessary to prevent regrowth . Growth delay as a function of dose rate Figure 3a -- e shows growth delays versus the initial dose rate after 1 , 3 or 7 days of continuous low dose rate exposure . The growth delay curves in Fig. 3a show that the radioresistant U-118MG cells had growth delays , after 24-h exposure , which increased slowly as a function of the initial dose rate . A growth delay of 2 months was obtained at about 0.7 , 0.3 and 0.2 Gy/h after 1 , 3 and 7 days of continuous exposure , respectively . The growth delay curves for the more radiosensitive U-373MG cells increased more steeply -LRB- Fig. 3b -RRB- , and a growth delay of 2 months was obtained at about 0.5 and 0.2 Gy/h after 1 and 3 days of continuous exposure , respectively . Thus , the U-373MG cells were more sensitive than the U-118MG cells also with regard to growth delay . Fig. 3Growth delay versus the initial dose rate after 1 , 3 or 7 days of continuous low dose rate exposure for a U-118MG , b U-373MG , c HT-29 , d A-431 and e SKBR-3 cells . The boxes at the top of each figure indicate at which dose rates and exposure times there was no regrowth -LRB- infinite growth delay or total kill of the cultures -RRB- . Mean values and maximal variations from four samples are shown Both the HT-29 cells -LRB- Fig. 3c -RRB- and the SKBR-3 cells -LRB- Fig. 3e -RRB- had a biphasic growth delay curve after 1 day 's exposure . The A-431 cells -LRB- Fig. 3d -RRB- had a slow increase in growth delay as a function of dose rate after 1 day 's exposure . The SKBR-3 cells had a growth delay that increased rather steeply as a function of the dose rate and reached a value of 2 months at about 0.50 and 0.15 Gy/h after 1 and 7 days of continuous exposure , respectively -LRB- Fig. 3e -RRB- . Growth delay as a function of total dose The same growth delay data as in Fig. 3a -- e are instead plotted as a function of the delivered total dose in Fig. 4a -- c , but here , for clarity , without the maximal variations . In all cases , there was a general increasing growth delay as a function of dose . Growth delays in the order of 10 days were obtained for all cells after delivery of total doses in the range 5 -- 15 Gy , independent of whether the dose was given during 1 , 3 or 7 days . Delays around 100 days required total doses of about 15 -- 20 Gy when given during 1 day and 25 -- 40 Gy when given during 3 or 7 days , for all cells . Fig. 4Growth delay versus total dose after a 1 , b 3 or c 7 days of continuous low dose rate exposure for U-118MG , U-373MG , HT-29 , A-431 and SKBR-3 cells . The growth delay values are the same as in Fig. 3 but , for clarity , without maximal variations Discussion The obtained results can hopefully serve as a guideline for the combinations of dose rate and exposure time necessary to kill tumour cells when applying low dose rate beta irradiation . The shift from regrowth to `` cure '' -LRB- no surviving cells -RRB- fell , for each cell type , within a narrow range of combinations . The U-118MG cells were more resistant to the treatments than the U-373MG and SKBR-3 cells . The A-431 and HT-29 cells showed intermediate resistance . However , the differences between the cell lines were not dramatic , and it can be generally stated that when applying 7 days of continuous irradiation , initial dose rates of about 0.2 -- 0.3 Gy/h were enough to kill all cells in the cultures . When exposed for 3 days , an initial dose rate in the order of 0.4 -- 0.6 Gy/h was needed . When the cells were exposed for only 24 h it was not possible to kill all the cells -LRB- with the exception of SKBR-3 cells -RRB- , even if the initial dose rate was as high as about 0.8 Gy/h . The studied dose rates are the highest that can be achieved in targeted radionuclide therapy -LSB- 10 , 16 , 18 -RSB- . The total doses achieved after 1 , 3 or 7 days ' exposure also correspond to the highest achievable doses in targeted radionuclide therapy -LSB- 16 -RSB- , and most often total doses of no more than 10 -- 20 Gy are obtained in targeting of B-cell lymphomas -LSB- 19 -RSB- . However , there are indications from preclinical studies that dramatic `` dose amplification '' per receptor interaction can be achieved by using effective residualising agents -LSB- 45 -RSB- . The obtained dose rates in beta particle-based radionuclide therapy are to a large extent a consequence not only of the amount of radionuclides bound to each tumour cell , but also of the cross-fire effect . This means that radionuclides bound to one cell also irradiate nearby cells owing to the long range of the radiation -LSB- 44 , 46 -RSB- . This can increase the dose rate tenfold , and makes it reasonable to assume that dose rates in the range used in our model experiments can be achieved in tumours in patients . The dose rate will be lower for a single tumour cell considering only the radionuclides bound to that cell -LSB- 25 -RSB- . Beta particles with a long range will permit rather uniform dose distributions and hopefully deliver therapeutically relevant radiation doses also to non-targeted tumour cells . Lower dose rates than we applied will probably not lead to curative treatments when beta particles are applied . From the obtained results it is obvious that , at least for the five types of tumour cell tested , we succeeded in finding `` dose rate -- exposure time '' combinations that could distinguish between `` cure '' -LRB- killing of 105 tumour cells -RRB- and `` relapse '' -LRB- recovery of tumour cells -RRB- . There may be cases in which only a fraction of the tumour cells have to be killed directly by radiation , since the remaining tumour cells may be killed through bystander effects -LSB- 30 -- 32 -RSB- or other factors -LRB- e.g. limited nutrition supply , natural immune response , adjuvant chemo - or immunotherapy -RRB- . However , our model can be applied under the assumption that 105 tumour cells have to be killed by radiation , even if other tumour cells are killed by other means . When considering radionuclide therapy it is , of course , also important to consider unwanted effects on normal tissues . The tolerance doses for most normal tissues are not known when exposure is to low dose rate irradiation . Targeted radionuclide therapy , using , for example , radiolabelled antibodies , fragments of antibodies or various receptor ligands , is expected to result in highly tumour-specific uptake of the therapeutic radionuclides . Thus , for a curative intent it is reasonable to establish the necessary tumour dose rates and exposure times . Another obvious question is which targeting agent should be tried for each type of tumour and , most importantly , whether the required conditions can be achieved without excessively severe side-effects on normal tissues . However , analyses of effects on normal tissues were beyond the scope of this study . Here we discuss the observed cell type-dependent differences . In a previous study -LSB- 35 -RSB- we published data on low dose rate acute effects , using three of the cells in the present study : U-118MG , U-373MG and HT-29 . In that study the initial dose rate was only 0.05 -- 0.09 Gy/h and the exposure time , 7 days . As would be expected in view of our new data , all cultures did grow after such treatment . It was shown that the U-373MG cells had , at day 7 , the most pronounced reduction in cell number owing to a combination of a G2 block and radiation-induced apoptosis . There were surprisingly low reductions in U-118MG and HT-29 cell numbers . U-118MG had a G2 block but no radiation-induced apoptosis . HT-29 had both a G2 block and some radiation-induced apoptosis , but the amount of apoptosis was smaller than for U-373MG cells . Thus , the results from that study indicated the U-373MG cells to be more sensitive than the other two cell lines owing to a higher degree of apoptosis . This is in agreement with the cell-killing results from the present study . The intrinsic radiosensitivity measured as S2Gy , after exposure to a high dose rate -LRB- most often 0.5 -- 2.0 Gy/min -RRB- photons has previously been determined for four of our studied cell lines . No determination of S2Gy for SKBR-3 cells was found in the literature . The results are given in Table 1 -LSB- 28 , 47 -- 54 -RSB- . Table 1Survival at 2Gy after exposure to high dose ratesCell lineSurvival at 2GyReferencesU-118MG0.44 -- 0.70 -LSB- 28 -RSB- , -LSB- 47 -RSB- U-373MG0.60 -- 0.62 -LSB- 28 -RSB- , -LSB- 48 -RSB- , -LSB- 49 -RSB- HT-290.55 -- 0.78 -LSB- 28 -RSB- , -LSB- 50 -RSB- , -LSB- 51 -RSB- , -LSB- 52 -RSB- , -LSB- 53 -RSB- , -LSB- 54 -RSB- A-431 ≈ 0.52 -LSB- 53 -RSB- There is also a review article on the intrinsic radiosensitivity , measured as cell survival at the dose 2Gy , S2Gy , for 694 human cell lines , of which 271 were from tumours -LSB- 55 -RSB- . The tumour cell lines were grouped according to tumour type , and the S2Gy values for the U-118MG and U-373MG gliomas and the A-431 cervical carcinoma used in this study fell within the expected range of values for the corresponding types of tumour . Thus , these cells can be considered typical for their tumour groups , at least regarding intrinsic radiosensitivity at high dose rate exposure . The HT-29 colorectal carcinoma is an exception , since it seems to be somewhat more radioresistant than most other colorectal carcinomas . Considering the values in Table 1 , it is clear that there is no relation between S2Gy and the effects of low dose rate irradiations . For example , the U-118MG cells were found to be most radioresistant to a low dose rate , while their S2Gy values were in the same range as for the other cell lines . The U-373MG cells , which were considered most sensitive to a low dose rate , also had S2Gy values in the same range . One possible explanation of the lack of agreement between intrinsic radiosensitivity measured as S2Gy and the effect of a low dose rate is that there are cell type-dependent differences in repopulation during low dose rate irradiations . Such differences can probably `` overshadow '' the differences in intrinsic radiosensitivity . Another possible explanation might be cell type-dependent differences in the capacity for low dose rate-induced apoptosis . The latter is supported by our previous study -LSB- 35 -RSB- , which showed that low dose rate-induced apoptosis was more frequent in U-373MG cells than in HT-29 cells and that no such apoptosis could be observed for U-118MG cells . Furthermore , it seems as if differences in hyperradiosensitivity -LRB- measured at low doses but at a high dose rate -RRB- are not of great importance , since the U-118MG and HT-29 cells have been reported to show hyperradiosensitivity , while U-373MG cells do not -LSB- 28 -RSB- . Had hyperradiosensitivity been of importance under the exposure conditions in this study , then U-373MG cells should have been more resistant than HT-29 and U-118MG cells . It has recently been suggested that variations in radiosensitivity at low dose rates are related to the compactness of chromatin -LSB- 56 -RSB- , but it is not known whether the cells in our study have any differences in this respect . In another recent experimental study , a good therapeutic effect at a low dose rate was reported ; in fact , if the total delivered dose was in the range 1 -- 2 Gy , the effect was as good as that achieved at a high dose rate , although the difference in dose rate was nearly three orders of magnitude -LSB- 57 -RSB- . This indicates that there are basic radiation biology aspects of low dose rate radiation that have to be analysed in much more detail . A clue to the molecular factors involved came from a recent report showing that activation or inhibition of the DNA damage sensor ATM is of importance -LSB- 58 -RSB- . It was found that DNA damage inflicted at a low rate failed to activate ATM ; however , if ATM was activated by chloroquine , the cells survived the low dose rate much better . We conclude that dose rates in the range 0.2 -- 0.3 Gy/h are necessary in order to kill 105 tumour cells during 1 week 's exposure . Higher dose rates , such as 0.4 -- 0.6 Gy/h and > 0.8 Gy/h , are necessary if the exposure times are only 3 days and 1 day , respectively . If , in some cases , a good therapeutic effect can not be obtained with beta particle radionuclide therapy because of too low dose rates and/or too short exposure times , then repeated -LRB- fractionated -RRB- treatment is a possibility to improve the result . However , the cellular response to repeated low dose rate exposures has to be analysed in further studies , since there is a risk of radiation-induced changes in the growth pattern of the tumour cells surviving the first exposure , as shown in Fig. 1b .

O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B I I O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O B I I O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B I I O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

low dose rate
radionuclide
tumour therapy


Functional sex differences in human primary auditory cortex Background We used PET to study cortical activation during auditory stimulation and found sex differences in the human primary auditory cortex -LRB- PAC -RRB- . Regional cerebral blood flow -LRB- rCBF -RRB- was measured in 10 male and 10 female volunteers while listening to sounds -LRB- music or white noise -RRB- and during a baseline -LRB- no auditory stimulation -RRB- . Introduction Previously , investigators tried to relate sex differences in behaviour and cognition to differences in brain anatomy , function , or connectivity . A well-documented example of sex differences in the brain is the difference in overall brain size . We now know that the size of the male brain is on average 8 -- 10 % larger than the female brain -LSB- 1 -- 3 -RSB- . It is also thought that , compared to the female brain , there is more hemispheric asymmetry in the anatomy of the male brain -LSB- 4 -- 7 -RSB- , which is supported by the finding that in the male brain , functions are represented more unilaterally -LSB- 8 -- 12 -RSB- . Sexual dimorphism has also been reported for the anatomy and function of the auditory cortex . The human auditory cortex is located in the superior temporal lobe . The primary auditory cortex -LRB- PAC -RRB- is situated in the medial two-thirds of the transverse temporal gyrus , also called Heschl 's gyrus -LRB- Fig. 1 -RRB- . It is surrounded by secondary and associative auditory regions , which cover the lateral part of the transverse temporal gyrus and extend to the superior temporal plane . The primary auditory region was designated as area 41 by Brodmann -LSB- 13 -RSB- , the secondary auditory regions are areas 42 and 22 -LSB- 14 -RSB- . An example of sexual dimorphism in the anatomy of the auditory system is in the study by Rademacher et al. -LSB- 15 -RSB- who reported that both left and right primary auditory cortices are larger in females than in males , although an earlier study found no differences -LSB- 7 -RSB- . Anatomical reports also show larger and more symmetrical auditory association cortices in females than in males -LSB- 7 , 16 -RSB- . However , many studies emphasize that there is intersubject variability regarding the size and location of the primary and secondary auditory cortex -LSB- 17 -- 21 -RSB- . Fig. 1Spatial distribution of significant increases in brain activation in men and women when comparing the auditory processing of noise , music , and a baseline . Activations are superimposed on an anatomical MRI template of SPM2 . Clusters are significant at p < 0.05 FDR corrected for multiple comparisons . L = left hemisphere , y = − 20 and y = − 6 means a coronal plane , respectively , 20 mm and 6 mm posterior to the anterior commissure , z = 8 means a horizontal plane 8 mm dorsal to the anterior commissure , z = − 1 means a horizontal plane 1 mm ventral to the anterior commissure . a Contrasting music with noise , women showed activation in the secondary auditory areas only , whereas men showed activation in both PAC and secondary auditory areas . b Comparing music to the baseline , both women and men showed bilateral activation in the PAC and secondary auditory areas . c Comparing noise to the baseline , women showed bilateral activation in the PAC . In men , on the other hand , no significant activation was found . The differences between men and women in a and c are significant -LRB- see Table 2 -RRB- Functional sex differences in auditory processing are widely studied in the light of language . Females depend less on their left hemisphere for language processing than males in some studies -LSB- 22 -RSB- , whereas in other studies the opposite was found -LSB- 23 , 24 -RSB- or no sex difference could be detected -LSB- 25 , 26 -RSB- . Whether a sex difference in language processing can be detected might depend on the nature of the task -LSB- 10 -RSB- . Sex-based influence on activation patterns in auditory regions is also found when manipulating the amount of background noise -LSB- 27 -RSB- , and auditory working memory tasks induce different activation patterns in males and females -LSB- 28 -RSB- . These studies suggest that combining men and women in auditory neuroimaging studies may obscure or bias results . However , most of these studies focus mainly on functional sex differences in higher order -LRB- associative -RRB- brain areas , thereby ignoring possible sex differences in primary auditory cortices . Previously , a sex difference in activation of the PAC during lipreading was found -LSB- 29 -RSB- . However , it has not been investigated whether nonspeech sounds can induce different activity in the PAC in both sexes . In the present study , we investigated potential sex differences in the activity of the primary auditory region , using different auditory stimuli . Material and methods Subjects Twenty healthy , right-handed volunteers participated in this study -LRB- 10 males , 10 females -RRB- . The mean age for men was 23 years -LRB- range 20 -- 25 -RRB- and for women 22 years -LRB- range 19 -- 27 -RRB- . All volunteers gave written informed consent according to the Declaration of Helsinki and the Medical Ethics Committee of the University Medical Center Groningen approved the study . None of the subjects had any history of psychiatric or neurological disorders . Prior to the scanning , subjects were tested for their hearing thresholds using standard audiometric measures . All subjects had normal hearing thresholds -LRB- < 20 dBHL , 0.25 -- 8 kHz -RRB- , and only small intersubject variations in hearing thresholds were observed . Data acquisition Regional cerebral blood flow -LRB- rCBF -RRB- was measured using radioactive water -LRB- -LSB- 15O -RSB- - water , half-life 122 s -RRB- as a tracer . A Siemens Ecat Exact HR+PET scanner , operated in three-dimensional mode with a 15.5 cm axial field of view , acquired 63 slices simultaneously . Each subject was scanned 12 times to measure the distribution of -LSB- 15O -RSB- - water with a 10 minute interval between two scans to allow for decay . Each scan was performed after an intravenous bolus injection of 500 MBq of -LSB- 15O -RSB- - water per scan . Except for the first scan , scanning started 30 s prior to injection , to account for background activation . Scanning continued for 120 seconds . The activity measured during this period was summed and used as a measure of rCBF . A scan specific calculated attenuation correction was performed to minimize interscan displacement-induced variance -LSB- 30 -RSB- . All subjects were scanned at fixed times on fixed weekdays , and male and female subjects were addressed to the scan dates randomly . Experimental design Three conditions were used in this study : baseline -LRB- no auditory stimulation -RRB- , white noise -LRB- at an intensity level of 75 dBSL -RRB- , and music -LRB- music of the movie `` The Piano '' at 75 dBSL -RRB- . Both music and noise have a wide frequency range , stimulating a large number of haircells in the cochlea and hence a large portion of the cortical auditory areas , but noise has a continuous and uniform frequency spectrum whereas music is a dynamic stimulus . Each condition was presented four times in a random order . For stimulus presentation we used a clinical audiometer -LRB- Interacoustics , model AC30 -RRB- , a Tandberg Educational tape recorder , and E.A.RTone 3A insert phones -LRB- with E.A.Rlink eartips -RRB- , which have a flat frequency response between 100 and 4000 Hz , measured in a Zwislocki-coupler . Stimuli were presented binaurally . Ten seconds before injection of radioactive water , the stimulus was started . Because the tracer reaches the brain approximately 10 seconds after injection , subjects were exposed to the stimulus for 20 seconds before the distribution of -LSB- 15O -RSB- - water in the brain starts . Subjects were instructed to close their eyes , not to move during the scans , and to listen to the auditory stimuli . Before each scan we informed the volunteers that the scan was about to start . Immediately after each scan the volunteers were questioned about the scan -LRB- did they hear the stimulus ? were they uncomfortable or distracted ? -RRB- . During scanning we monitored the subjects with infrared cameras . Data analysis The 2002 version of Statistical Parametric Mapping -LRB- SPM2 : software from the Wellcome Department of Cognitive Neurology , London , UK -RRB- was used for spatial transformations -LRB- realignment , transformation into standard stereotactic space , and smoothing with an isotropic Gaussian kernel of 8 mm FWHM -RRB- , and statistical analysis -LSB- 31 -RSB- . An ANOVA estimated the following parameters : two groups -LRB- male and female -RRB- , three conditions -LRB- baseline , noise , and music -RRB- and the mean perfusion to normalize for global flow differences -LRB- multigroup , conditions , and covariates -RRB- . Each scan was scaled to a mean global activity of 50 ml/100 ml/min . Hypotheses about regionally specific condition effects were tested to compare the estimates by using linear compounds or contrasts . The resulting set of voxel values for these contrasts constituted the associated SPM of the t-statistics . The significance threshold used for the analysis of the two groups separately -LRB- male -- female -RRB- was p < 0.05 -LRB- false discovery rate -LRB- FDR -RRB- corrected for multiple comparisons -LSB- 32 -RSB- -RRB- with an extent cluster threshold of more than 8 voxels . We used AMIDE software -LRB- http://amide.sourceforge.net/ -RRB- for colour scaling and display of the results on the anatomical MRI template of SPM2 . For maximum statistical sensitivity and for testing the significance of the sex related differences , we conducted a region of interest -LRB- ROI -RRB- analysis in our a priori hypothesized areas , i.e. the left and right PAC , using the SPM anatomy toolbox -LSB- 33 -RSB- and the MarsBaR toolbox -LRB- MARSeille Boîte À Région d'intérêt -LSB- 34 -RSB- -RRB- . Specifically , we created anatomical ROIs based on the three-dimensional probabilistic cytoarchitectonic maps from the SPM anatomy toolbox brains -LSB- 18 , 20 , 33 -RSB- . To compensate for differences in stereotactic space between SPM and the anatomy toolbox , a linear transformation was applied to the anatomical ROIs . MarsBaR was then used to conduct the statistical analyses on these ROIs . The statistical procedure in MarsBaR is the same as in SPM , but instead of analysing on a voxel-by-voxel basis like SPM does , all voxels in a region are averaged , and hence inferences about the whole region can be made . Also in MarsBaR , contrasts were considered significant at p < 0.05 . Results Music versus noise For this contrast , in females the SPM-analysis resulted in significant bilateral activation clusters with a maximal significant voxel in the secondary auditory areas . In men much larger bilateral clusters were found covering not only the secondary auditory areas but also the PAC -LRB- Fig. 1a and Table 1 -RRB- . This indicates that males have a much larger activation in the PAC during music than during noise . The voxel-wise analysis of SPM did not reveal any significant activation differences between the two sexes . Table 1Overview of brain areas with statistically significant cerebral blood flow changes SideRegionTalairach coordinatesNumber of voxelst-valuexyzMusic vs noiseFemalesRightBA 2261 − 6 − 11,4597.77 LeftBA 22 − 51 − 10 − 13105.02 MalesRightBA 41 , 42 , 22522 − 33,2347.87 LeftBA 41 , 42 , 22 − 51 − 802,1907.87 Music vs baselineFemalesRightBA 41 , 42 , 2257 − 4 − 12,7508.85 LeftBA 41 , 42 , 22 − 48 − 1731,8667.97 MalesRightBA 41 , 42 , 2251 − 1022,47510.36 LeftBA 41 , 42 , 22 − 48 − 1212,3068.67 Noise vs baselineFemalesRightBA 4146 − 2154466.49 LeftBA 41 − 40 − 2354636.24 MalesNo suprathreshold clustersBaseline vs noiseFemalesNo suprathreshold clustersMalesRightBA 92050299685.20 Baseline vs musicMalesNo suprathreshold clustersFemalesNo suprathreshold clustersThe region , covered by the whole cluster and noted in Brodmann areas , and the number of voxels in the cluster are described -LSB- 14 -RSB- . BA 41 corresponds to the PAC , BA 22 and 42 correspond to secondary auditory areas . Only the stereotaxic -LRB- Talairach -RRB- coordinates and t-value of the maximum of the cluster are given . The significance threshold was p < 0.05 FDR-corrected for multiple comparisons ; extent threshold was 8 voxels ; voxel size was 2 × 2 × 2 mm . Brain regions were identified using the Talairach atlas and the stereotactic atlas of the human brain of Mai et al. -LSB- 52 , 53 -RSB- The region of interest analysis in the PAC showed that females do have a larger activation in the PAC during music than during noise -LRB- p values 0.005 and 0.001 for the left and right PAC , respectively -RRB- . But the difference between music and noise is much smaller than in males in both the left and right PAC -LRB- p values 0.016 and 0.008 , respectively , Table 2 , Fig. 2 -RRB- . No significant deactivations were found -LRB- i.e. noise versus music -RRB- . Table 2Region of interest analysis of the left and right primary auditory cortex -LRB- PAC -RRB- P-valueLeft PACRight PACMusic vs noise females0 .005 * 0.001 * Males < 0.001 * < 0.001 * Females minus males0 .9840.992 Males minus females0 .016 * 0.008 * Music vs baseline females < 0.001 * < 0.001 * Males < 0.001 * < 0.001 * Females minus males0 .7210.779 Males minus females0 .2790.221 Noise vs baseline females < 0.001 * < 0.001 * Males0 .016 * 0.005 * Females minus males0 .042 * 0.034 * Males minus females0.9580.966One-sided statistical significance for various contrasts was tested for men and women separately as well as for the differences between both . * Significant at p < 0.05 Fig. 2Regional cerebral blood flow relative to the baseline of each group , based on all voxels in the left and right PAC -LRB- with a global mean flow of 50 ml/100 g/min -RRB- . Error bars indicate the 90 % confidence interval of the mean across subjects per condition ; the confidence interval of the baseline is also given To determine whether this sex difference can be attributed to the processing of either music or noise , we compared these two stimuli with a baseline without experimental auditory stimulation . Music versus baseline For this contrast , the SPM-analysis showed large comparable activation clusters covering primary and secondary areas in both males and females -LRB- Fig. 1b and Table 1 -RRB- . The ROI analysis showed that there were no significant differences in the activation of the PAC between both sexes for this contrast -LRB- Table 2 , Fig. 2 -RRB- . Again , no significant deactivations were found -LRB- baseline versus music -RRB- . Noise versus baseline Comparing noise to the baseline , the SPM-analysis showed two significant bilateral activation clusters with maxima in the PAC for the female group . In contrast , no significant activations were found in the male brain at a corrected level of p < 0.05 -LRB- Fig. 1c and Table 1 -RRB- . Only when the data were analysed at an uncorrected threshold of p < 0.01 , a small activation appeared in the PAC of males , primarily on the right side -LRB- Fig. 3 -RRB- . The ROI-analysis confirmed in men the involvement of the PAC while processing noise , showing significant bilateral increases of blood flow , but these activations were significantly less than in women in the left -LRB- 0.042 -RRB- and right PAC -LRB- p = 0.034 -RRB- -LRB- Table 2 -RRB- . Fig. 3Spatial distribution of significant increases in brain activation in men and women for the comparison of noise to the baseline . Clusters are significant at p < 0.01 , uncorrected for multiple comparisons . L = left hemisphere , y = − 20 means a coronal plane 20 mm posterior to the anterior commissure , z = 8 means a horizontal plane 8 mm dorsal to the anterior commissure . In contrast to Fig. 1c , at an uncorrected level men do show activation in PAC , but it is much smaller than in the female group The voxel-wise analysis of SPM also revealed a significant deactivation in the male group . This deactivation was located in the right dorsolateral part of the prefrontal cortex extending to the posterior part of the middle frontal gyrus , covering primarily BA 9 -LRB- Fig. 4 and Table 1 -RRB- . In contrast , no significant deactivation was found in the female group . Fig. 4Spatial distribution of significant decreases in brain activation in men and women for the comparison of noise to the baseline . Clusters are significant at p < 0.05 , FDR corrected for multiple comparisons . x = 20 means a sagittal plane 20 mm on the right of the anterior commissure , z = 29 means a horizontal plane 29 mm dorsal to the anterior commissure . Only men showed a significant deactivation in the dorsolateral prefrontal cortex Discussion Our data demonstrate a sex difference in regional cerebral blood flow in the left and right primary auditory cortex -LRB- PAC -RRB- when comparing auditory processing of music and noise . The PAC was more activated by music than by noise in both men and women . But this difference between the two stimuli was significantly higher in men than in women . To determine whether this sex difference can be attributed to the processing of music , noise , or both , we compared the two stimuli with a baseline of no auditory stimulation . Comparing music with the baseline resulted in extensive activation of the primary and secondary auditory cortex in both sexes , but no significant sex difference was found . On the other hand , a sex difference was detected in the processing of noise , because females activated their PAC significantly more than males . The finding that sex differences in auditory processing can already be detected at the level of the primary cortex is very important , because it is often assumed that sex differences act exclusively upon higher-order cortical areas . There appears to be a mismatch between the SPM and ROI analyses because there is a difference in power between the two analysing methods . The ROI analysis pools the data of all voxels in the PAC and gives a reduced standard error , resulting in a higher t-value and hence more power -LSB- 34 -RSB- . Although the SPM analysis showed no activation at all for the male group and a very significant activation for the female group , it did not have enough power to detect a significant difference between the two groups . Because of the enhanced sensitivity of an ROI analysis , this analysis did show a significant sex difference . Of course , caution has to be made when pooling data to improve the sensitivity and is therefore only allowed in a priori defined regions , in this case the PAC . The question is whether the sex difference found is caused by the experimental conditions or by other factors like anatomical differences or methodological errors . The reported differences in activation of the PAC stand or fall by a correct identification of this area in the region of interest analysis . Several authors reported substantial intersubject variability in size and location of the PAC -LSB- 17 -- 21 -RSB- . To overcome this problem of variability in size and location of the PAC , we employed linear resizing and shearing as well as nonlinear warping of the brain to normalize the individual brains into a standardized stereotactic frame . In addition , a Gaussian smoothing filter of 8 mm was used to remove residual variance in brain structure that remains after the stereotactic normalization . Furthermore , we used probability maps , based on the cytoarchitecture of 10 subjects , to identify the PAC -LSB- 18 , 20 , 33 -RSB- . Other studies suggested that the volume of the PAC is bilaterally larger in females than in males -LSB- 15 -RSB- . It is therefore necessary that the region of interest depicts the PAC of both men and women . This is the case , because the probability maps of the SPM anatomy toolbox are based on the cytoarchitecture of five male and five female brains -LSB- 18 , 20 , 33 -RSB- . For these reasons we believe that possible intersubject or intergroup differences in size and location of the PAC do not bias our results . If the reported sex and contrast dependent differences in rCBF patterns are not caused by underlying anatomical differences , metabolic differences , or methodological errors , they must have been induced by the experimental stimulus . During the baseline condition , subjects had to lie quietly in the scanner and no auditory stimulus was applied . Without auditory stimulation and a specific task , the variance in rCBF might increase , resulting in less statistical power when comparing conditions to the baseline . However , as shown in Fig. 2 , the confidence interval for the model parameters was very similar for the three conditions . In addition , the confidence interval was also very similar for the two sexes . This means that the different contrasts tested have similar statistical power . Considering the significant sexual dichotomy in the processing of music versus noise and the results when comparing either the music or noise with the baseline condition , we conclude that males and females differ in the processing of noise . Thus our data demonstrate a sexual dichotomy in auditory processing . But which mechanism could explain this sexual dichotomy ? A key to answering this question is the role of the prefrontal cortex , which we found to be deactivated in men when listening to noise . In humans , the prefrontal cortex is engaged in diverse cognitive processes including cognitive control , working memory , and attention -LSB- 35 -RSB- . For example , Gisselgård et al. -LSB- 36 , 37 -RSB- investigated the influence of irrelevant speech on working memory tasks and revealed a functional link between auditory and prefrontal regions . Tzourio et al. demonstrated that prefrontal areas are engaged in auditory tasks that involve sustained or selective auditory attention -LSB- 38 -RSB- . In the present study , no explicit -LRB- attention -RRB- task was implemented . Subjects lay passively in the scanner and were instructed to listen to the auditory stimuli . While listening to an insignificant stimulus like noise , males deactivated the prefrontal attention areas as compared to silence . Females , on the other hand , had no deactivation of the attention areas and had a higher activation in the primary auditory cortex . Deactivation of the prefrontal regions was only seen in the right hemisphere , which is consistent with Tzourio et al. who stated that a right hemisphere dominance exists for attention -LSB- 38 -RSB- . The present results suggest a relationship between activation of PAC and prefrontal cortex . From literature , anatomical evidence exists concerning auditory -- prefrontal connections . Studies on monkeys have shown that the prefrontal cortex is reciprocally connected with auditory association areas -LRB- i.e. belt and parabelt in the monkey brain -RRB- -LSB- 39 -RSB- . The secondary and primary auditory areas are reciprocally connected -LRB- c.f. -LSB- 40 -RSB- -RRB- . More recent studies on monkeys identified two auditory -- prefrontal processing streams : dorso and ventrolateral auditory streams -LSB- 41 -- 43 -RSB- . Although , one should be cautious when comparing human and nonhuman primate brains , a similar organization of several parts of the human and monkey prefrontal cortex has been reported -LSB- 44 , 45 -RSB- . To summarize , we know from literature that the auditory and prefrontal regions are anatomically and functionally linked , and our data show a sexual dichotomy in the -LRB- de -RRB- activation of both regions . Apparently , the male and female brains handle an insignificant stimulus like noise differently , and we speculate that this is done by a different engagement of the auditory -- prefrontal attention network . Namely , differences in attention result in a different deactivation of the right prefrontal cortex , which in turn modulates the activation of the PAC and thus explains the sex differences found in the activation of the PAC . This corresponds with previous findings that sex differences exist in the frontal -- temporal network , namely , males have higher intrahemispheric functional connectivity of frontal and temporal areas than females -LSB- 46 -RSB- . It is also known from ERP studies that sex differences exist in orienting attention to auditory stimuli -LSB- 47 -RSB- . It must be noted that even though our data indicate differences in the auditory system , no independent behavioural data regarding the attention levels during scanning are available . Hence , the present experimental design only allows us to speculate about the correlation between the different deactivation of the prefrontal cortex and differences in attention . Further research is needed to fully clarify the role of attention on PAC activation and to determine whether our results can be repeated . Furthermore , other auditory conditions like pure tones , noise bursts , and speech -LRB- sense and nonsense -RRB- sounds are needed to fully map the influence of sex on central auditory processing and to investigate lateralization differences between the sexes . To our knowledge , this is the first time that a sexual dichotomy in the function of the PAC is demonstrated using nonspeech sounds . Previously , a sex difference in the PAC was demonstrated while lip reading -LSB- 29 , 48 -RSB- . These studies showed only female activation in the PAC during lip reading because they associate the absent speech sound with the visual lip movements . Males on the other hand , did not display activation in the PAC and focus on the present visual lip image itself . The current study shows that even simple sounds induce different activation patterns , especially in the PAC . Other sexual dichotomies in the central auditory system have been reported . For example , sex differences in auditory feedback loops of the corticofugal network have been found in which men showed more suppression of repeated acoustic stimuli than females . This sex difference in auditory gating is probably the result of differential neuronal inhibition to repeated stimulation -LSB- 49 -RSB- . Likewise , the stronger spontaneous otoacoustic emissions -LRB- SOAEs -RRB- in females are thought to originate from a relatively larger amount of efferent inhibition in males -LSB- 50 -RSB- . This efferent innervation would start in the olivary complex and terminate in the outer hair cells of the cochlea -LSB- 50 -RSB- . Although the present sexual dichotomy would be consistent with the concept of evolutionary advantages in a hunter -- gatherer society -LRB- c.f. -LSB- 51 -RSB- -RRB- where the inhibition of constant irrelevant stimuli in men may facilitate them to focus their attention to a single task , e.g. hunting , this concept remains speculation . In conclusion , a very significant sexual dichotomy was found in the activation of the PAC with different types of acoustic stimuli -LRB- noise and music -RRB- together with sex differences in deactivation of prefrontal areas . It is known that the auditory and prefrontal regions are anatomically and functionally linked , and the prefrontal cortex is engaged in auditory attention tasks . Hence , we hypothesize that differences in attention might result in a different involvement of the right prefrontal cortex , which in turn modulates the activation of the PAC . This shows that sex influences brain activity already at the level of primary sensory cortex and that in functional imaging studies on primary sensory cortical areas , sex can not be ignored .

O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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