Potential benefit of MRI-guided IMRT for flank irradiation in pediatric patients with Wilms' tumor.

PURPOSE/OBJECTIVE: Flank irradiation for Wilms' tumor (WT) is currently performed at our institute using a cone-beam computed tomography-guided volumetric modulated arc (VMATCBCT) workflow. By adding real-time magnetic resonance imaging (MRI) guidance to the treatment, safety margins could be reduced. The study purpose was to quantify the potential reduction of the planning target volume (PTV) margin and its dosimetric impact when using an MRI-guided intensity modulated radiation therapy (IMRTMRI) workflow compared to the VMATCBCT workflow. MATERIAL/METHODS: 4D-CT, MRI and CBCT scans acquired during preparation and treatment of 15 patients, were used to estimate both geometric, motion and patient set-up systematic (∑) and random (σ) errors for VMATCBCT and IMRTMRI workflows. The mean PTV (PTVmean) expansion was calculated using the van Herk formula. Treatment plans were generated using five margin scenarios (PTVmean ± 0, 1 and 2 mm). Furthermore, the IMRTMRI plans were optimized with a 1.5T transverse magnetic field turned-on to realistically model an MRI-guided treatment. Plans were evaluated using dose-volume statistics (p<.01, Wilcoxon). RESULTS: Analysis of ∑ and σ errors resulted in a PTVmean of 5 mm for the VMATCBCT and 3 mm for the IMRTMRI workflows in each orthogonal direction. Target coverage was unaffected by the margin decrease with a mean V95%=100% for all margin scenarios. For the PTVmean, an average reduction of the mean dose to the organs at risk (OARs) was achieved with IMRTMRI compared to VMATCBCT: 3.4 ± 2.4% (p<.01) for the kidney, 3.4 ± 2.1% (p<.01) for the liver, 2.8 ± 3.0% (p<.01) for the spleen and 4.9 ± 3.8% (p<.01) for the pancreas, respectively. CONCLUSIONS: Imaging data in children with WT demonstrated that the PTV margin could be reduced isotropically down to 2 mm when using the IMRTMRI compared to the VMATCBCT workflow. The former results in a dose reduction to the OARs while maintaining target coverage.

Sparing the surgical area with stereotactic body radiotherapy for combined treatment of spinal metastases: a treatment planning study.

INTRODUCTION: Decreasing the radiation dose in the surgical area is important to lower the risk of wound complications when surgery and radiotherapy are combined for the treatment of spinal metastases. The purpose of this study was to compare the radiation dose in the surgical area for spinal metastases between single fraction external beam radiotherapy (EBRT), single fraction stereotactic body radiotherapy (SBRT) and single fraction SBRT with active sparing (SBRT-AS) of the posterior surgical area. METHODS: Radiotherapy treatment plans for EBRT, SBRT and SBRT-AS of the posterior surgical area were created for 13 patients with spinal metastases. A single fraction of 8Gy was prescribed to the spinal metastasis in the EBRT plan. For the SBRT treatment plans, a single fraction of 18Gy was prescribed to the metastasis and 8Gy to the rest of the vertebral body. For the SBRT plan with active sparing the dose in the designated surgical area was minimized without compromising the dose to the organs at risk. RESULTS: The median dose in the surgical area was 2.6Gy (1.6-5.3Gy) in the SBRT plan with active sparing of the surgical area compared to a median dose of 3.7Gy (1.6-6.3Gy) in the SBRT plan without sparing and 6.5Gy (3.5-9.1Gy) in the EBRT plans (p < .001). The radiation doses to the spinal metastases and organs at risk were not significantly different between the SBRT plan with and without sparing the surgical area. CONCLUSIONS: The radiation dose to the surgical area is significantly decreased with the use of SBRT compared to EBRT. Active sparing of the surgical area further decreased the mean radiation dose in the surgical area without compromising the dose to the spinal metastasis and the organs at risk.

Intra-fractional per-beam adaptive workflow to mitigate the need for a rotating gantry during MRI-guided proton therapy.

The integration of real-time magnetic resonance imaging (MRI) guidance and proton therapy would potentially improve the proton dose steering capability by reducing daily uncertainties due to anatomical variations. The use of a fixed beamline coupled with an axial patient couch rotation would greatly simplify the proton delivery with MRI guidance. Nonetheless, it is mandatory to assure that the plan quality is not deteriorated by the anatomical deformations due to patient rotation. In this work, an in-house tool allowing for intra-fractional per-beam adaptation of intensity-modulated proton plans (BeamAdapt) was implemented through features available in RayStation. A set of three MRIs was acquired for two healthy volunteers (V1,V2): (1) no rotation/static, (2) rotation to the right and (3) left.V1was rotated by 15°, to simulate a clinical pediatric abdominal case andV2by 45°, to simulate an extreme patient rotation case. For each volunteer, a total of four intensity-modulated pencil beam scanning plans were optimized on the static MRI using virtual abdominal targets and two-three posterior-oblique beams. Beam angles were defined according to the angulations on the rotated MRIs. With BeamAdapt, each original plan was initially converted into separate plans with one beam per plan. In an iterative order, individual beam doses were non-rigidly deformed to the rotated anatomies and re-optimized accounting for the consequent deformations and the beam doses delivered so far. For evaluation, the final accumulated dose distribution was propagated back to the static MRI. Planned and adapted dose distributions were compared by computing relative differences between dose-volume histogram metrics. Absolute target dose differences were on average below 1% and organs-at-risk mean dose differences were below 3%. With BeamAdapt, not only intra-fractional per-beam proton plan adaptation coupled with axial patient rotation is possible but also the need for a rotating gantry during MRI guidance might be mitigated.

Deep learning prediction of proton and photon dose distributions for paediatric abdominal tumours.

OBJECTIVE: Dose prediction using deep learning networks prior to radiotherapy might lead tomore efficient modality selections. The study goal was to predict proton and photon dose distributions based on the patient-specific anatomy and to assess their clinical usage for paediatric abdominal tumours. MATERIAL AND METHODS: Data from 80 patients with neuroblastoma or Wilms' tumour was included. Pencil beam scanning (PBS) (5 mm/ 3%) and volumetric-modulated arc therapy (VMAT) plans (5 mm) were robustly optimized on the internal target volume (ITV). Separate 3-dimensional patch-based U-net networks were trained to predict PBS and VMAT dose distributions. Doses, planning-computed tomography images and relevant optimization masks (ITV, vertebra and organs-at-risk) of 60 patients were used for training with a 5-fold cross validation. The networks' performance was evaluated by computing the relative error between planned and predicted dose-volume histogram (DVH) parameters for 20 inference patients. In addition, the organs-at-risk mean dose difference between modalities was calculated using planned and predicted dose distributions (ΔDmean = DVMAT-DPBS). Two radiation oncologists performed a blind PBS/VMAT modality selection based on either planned or predicted ΔDmean. RESULTS: Average DVH differences between planned and predicted dose distributions were ≤ |6%| for both modalities. The networks classified the organs-at-risk Dmean difference as a gain (ΔDmean > 0) with 98% precision. An identical modality selection based on planned compared to predicted ΔDmean was made for 18/20 patients. CONCLUSION: Deep learning networks for accurate prediction of proton and photon dose distributions for abdominal paediatric tumours were established. These networks allowing fast dose visualisation might aid in identifying the optimal radiotherapy technique when experience and/or resources are unavailable.

Morphological changes after cranial fractionated photon radiotherapy: Localized loss of white matter and grey matter volume with increasing dose.

PURPOSE: Numerous brain MR imaging studies have been performed to understand radiation-induced cognitive decline. However, many of them focus on a single region of interest, e.g. cerebral cortex or hippocampus. In this study, we use deformation-based morphometry (DBM) and voxel-based morphometry (VBM) to measure the morphological changes in patients receiving fractionated photon RT, and relate these to the dose. Additionally, we study tissue specific volume changes in white matter (WM), grey matter (GM), cerebrospinal fluid and total intracranial volume (TIV). METHODS AND MATERIALS: From our database, we selected 28 patients with MRI of high quality available at baseline and 1 year after RT. Scans were rigidly registered to each other, and to the planning CT and dose file. We used DBM to study non-tissue-specific volumetric changes, and VBM to study volume loss in grey matter. Observed changes were then related to the applied radiation dose (in EQD2). Additionally, brain tissue was segmented into WM, GM and cerebrospinal fluid, and changes in these volumes and TIV were tested. RESULTS: Performing DBM resulted in clusters of dose-dependent volume loss 1 year after RT seen throughout the brain. Both WM and GM were affected; within the latter both cerebral cortex and subcortical nuclei show volume loss. Volume loss rates ranging from 5.3 to 15.3%/30 Gy were seen in the cerebral cortical regions in which more than 40% of voxels were affected. In VBM, similar loss rates were seen in the cortex and nuclei. The total volume of WM and GM significantly decreased with rates of 5.8% and 2.1%, while TIV remained unchanged as expected. CONCLUSIONS: Radiotherapy is associated with dose-dependent intracranial morphological changes throughout the entire brain. Therefore, we will consider to revise sparing of organs at risk based on future cognitive and neurofunctional data.

Lasting biological effects of early environmental influences. II. Lasting depression of weight caused by neonatal contamination.

Certain specific-pathogen-free (SPF) mice bred and maintained under semiprotected conditions have an intestinal flora which is qualitatively simpler (although not quantitatively smaller) than that of mice of the same genetic stock produced under ordinary conditions. They are also heavier at weaning time, grow at a faster rate, and reach a greater adult weight than ordinary mice. When SPF mice are contaminated per os shortly after birth with certain bacterial cultures isolated from the intestinal contents of adult ordinary mice, these bacteria multiply extensively throughout the gastrointestinal tract and persist at extremely high levels until weaning time. Such bacterial infections do not affect significantly either weaning weight, growth rate, or maximum adult weight. In contrast, weight depression could be consistently brought about by contaminating newborn SPF mice per os with bacteria-free filtrates of homogenates of intestines from ordinary mice. The weight-depressing agent passed through Millipore discs of 0.45 and 0.22 micro porosity, but was held back at 0.10 micro porosity. The depression of weight caused by either intestine homogenate or filtrates thereof could be detected within a few days after contamination (of 2 day old mice) and persisted throughout the adult life of the contaminated animals. When intestine homogenate of the SPF mice used in this study were introduced per os into newborn SPF mice, they did not affect their growth rate or adult weight. On several occasions, but not consistently, bacteria-free filtrates capable of depressing the weight curve of SPF mice produced alterations in the appearance of tissue cultures of BHK-21 and mouse embryo cells. When tissue cultures so infected were introduced into newborn SPF mice, the weight of these animals was depressed early and lastingly. An agent exhibiting weight-depressing activity has been transferred from mouse to mouse over many passages by contaminating newborn SPF animals per os. Weight depression was achieved with extremely small doses of material (10(-5) ml of intestine homogenate of 10(-4) of mouse embryo culture). Under these conditions, none of the animals showed obvious signs of disease except reduced weight. Only very young SPF mice (preferably less than 3 days old) proved susceptible to the weight-depressing effect of the filtrates of intestine homogenates or of infected tissue cultures prepared therefrom. After oral contamination, it took approximately 1 wk before the intestinal homogenate obtained from contaminated animals exhibited a high level of weight-depressing activity. The growth-depressing effect could be transmitted from one generation to the next by mating SPF mice that had been contaminated shortly after birth and that were consequently smaller than control SPF animals.

2D dosimetry in a proton beam with a scintillating GEM detector.

A two-dimensional position-sensitive dosimetry system based on a scintillating gas detector is being developed for pre-treatment verification of dose distributions in particle therapy. The dosimetry system consists of a chamber filled with an Ar/CF(4) scintillating gas mixture, inside which two gas electron multiplier (GEM) structures are mounted (Seravalli et al 2008b Med. Phys. Biol. 53 4651-65). Photons emitted by the excited Ar/CF(4) gas molecules during the gas multiplication in the GEM holes are detected by a mirror-lens-CCD camera system. The intensity distribution of the measured light spot is proportional to the 2D dose distribution. In this work, we report on the characterization of the scintillating GEM detector in terms of those properties that are of particular importance in relative dose measurements, e.g. response reproducibility, dose dependence, dose rate dependence, spatial and time response, field size dependence, response uniformity. The experiments were performed in a 150 MeV proton beam. We found that the detector response is very stable for measurements performed in succession (sigma = 0.6%) and its response reproducibility over 2 days is about 5%. The detector response was found to be linear with the dose in the range 0.05-19 Gy. No dose rate effects were observed between 1 and 16 Gy min(-1) at the shallow depth of a water phantom and 2 and 38 Gy min(-1) at the Bragg peak depth. No field size effects were observed in the range 120-3850 mm(2). A signal rise and fall time of 2 micros was recorded and a spatial response of

Characterization of a scintillating GEM detector with low energy x-rays.

A two-dimensional position-sensitive dosimetry system based on a scintillating gas detector is being developed with the aim of using it for pre-treatment verification of dose distributions in charged particle therapy. The dosimetry system consists of a chamber filled with an Ar/CF(4) scintillating gas mixture, inside which two cascaded gas electron multipliers (GEMs) are mounted. A GEM is a thin kapton foil with copper cladding structured with a regular pattern of sub-mm holes. In such a system, light quanta are emitted by the scintillating gas mixture during the electron avalanches in the GEM holes when radiation traverses the detector. The light intensity distribution is proportional to the energy deposited in the detector's sensitive volume by the beam. In the present work, we investigated the optimization of the scintillating GEM detector light yield. The light quanta are detected by means of a CCD camera or a photomultiplier tube coupled to a monochromator. The GEM charge signal is measured simultaneously. We have found that with 60 microm diameter double conical GEM holes, a brighter light signal and a higher electric signal are obtained than with 80 microm diameter holes. With an Ar + 8% CF(4) volume concentration, the highest voltage across the GEMs and the largest light and electric signals were reached. Moreover, we have found that the emission spectrum of Ar/CF(4) is independent of (1) the voltages applied across the GEMs, (2) the x-ray beam intensity and (3) the GEM hole diameter. On the other hand, the ratio of Ar to CF(4) peaks in the spectrum changes when the concentration of the latter gas is varied.

A scintillating gas detector for 2D dose measurements in clinical carbon beams.

A two-dimensional position sensitive dosimetry system based on a scintillating gas detector has been developed for pre-treatment verification of dose distributions in hadron therapy. The dosimetry system consists of a chamber filled with an Ar/CF4 scintillating gas mixture, inside which two cascaded gas electron multipliers (GEMs) are mounted. A GEM is a thin kapton foil with copper cladding structured with a regular pattern of sub-mm holes. The primary electrons, created in the detector's sensitive volume by the incoming beam, drift in an electric field towards the GEMs and undergo gas multiplication in the GEM holes. During this process, photons are emitted by the excited Ar/CF4 gas molecules and detected by a mirror-lens-CCD camera system. Since the amount of emitted light is proportional to the dose deposited in the sensitive volume of the detector by the incoming beam, the intensity distribution of the measured light spot is proportional to the 2D hadron dose distribution. For a measurement of a 3D dose distribution, the scintillating gas detector is mounted at the beam exit side of a water-bellows phantom, whose thickness can be varied in steps. In this work, the energy dependence of the output signal of the scintillating gas detector has been verified in a 250 MeV/u clinical 12C ion beam by means of a depth-dose curve measurement. The underestimation of the measured signal at the Bragg peak depth is only 9% with respect to an air-filled ionization chamber. This is much smaller than the underestimation found for a scintillating Gd2O2S:Tb ('Lanex') screen under the same measurement conditions (43%). Consequently, the scintillating gas detector is a promising device for verifying dose distributions in high LET beams, for example to check hadron therapy treatment plans which comprise beams with different energies.

Characterization of a prototype MR-compatible Delta4 QA system in a 1.5 tesla MR-linac.

To perform patient plan quality assurance (QA) on a newly installed MR-linac (MRL) it is necessary to have an MR-compatible QA device. An MR compatible device (MR-Delta4) has been developed together with Scandidos AB (Uppsala, Sweden). The basic characteristics of the detector response, such as short-term reproducibility, dose linearity, field size dependency, dose rate dependency, dose-per-pulse dependency and angular dependency, were investigated for the clinical Delta4-PT as well as for the MR compatible version. All tests were performed with both devices on a conventional linac and the MR compatible device was tested on the MRL as well. No statistically significant differences were found in the short-term reproducibility (<0.1%), dose linearity (⩽0.5%), field size dependency (<2.0% for field sizes larger than 5 × 5 cm2), dose rate dependency (<1.0%) or angular dependency for any phantom/linac combination. The dose-per-pulse dependency (<0.8%) was found to be significantly different between the two devices. This difference can be explained by the fact that the diodes in the clinical Delta4-PT were irradiated with a much larger dose than the MR-Delta4-PT ones. The absolute difference between the devices (<0.5%) was found to be small, so no clinical impact is expected. For both devices, the results were consistent with the characteristics of the Delta4-PT device reported in the literature (Bedford et al 2009 Phys. Med. Biol. 54 N167-76; Sadagopan et al 2009 J. Appl. Clin. Med. Phys. 10 2928). We found that the characteristics of the MR compatible Delta4 phantom were found to be comparable to the clinically used one. Also, the found characteristics do not differ from the previously reported characteristics of the commercially available non-MR compatible Delta4-PT phantom. Therefore, the MR compatible Delta4 prototype was found to be safe and effective for use in the 1.5 tesla magnetic field of the Elekta MR-linac.

Inter-observer agreement in GTV delineation of bone metastases on CT and impact of MR imaging: A multicenter study.

BACKGROUND AND PURPOSE: The use of Stereotactic Body Radiotherapy (SBRT) for bone metastases is increasing rapidly. Therefore, knowledge of the inter-observer differences in tumor volume delineation is essential to guarantee precise dose delivery. The aim of this study is to compare inter-observer agreement in bone metastases delineated on different imaging modalities. MATERIAL AND METHODS: Twenty consecutive patients with bone metastases treated with SBRT were selected. All patients received CT and MR imaging in treatment position prior to SBRT. Five observers from three institutions independently delineated gross tumor volume (GTV) on CT alone, CT with co-registered MRI and MRI alone. Four contours per imaging modality per patient were available, as one set of contours was shared by 2 observers. Inter-observer agreement, expressed in generalized conformity index [CIgen], volumes of contours and contours center of mass (COM) were calculated per patient and imaging modality. RESULTS: Mean GTV delineated on MR (45.9±52.0cm3) was significantly larger compared to CT-MR (40.2±49.4cm3) and CT (34.8±41.8cm3). A considerable variation in CIgen was found on CT (mean 0.46, range 0.15-0.75) and CT-MRI (mean 0.54, range 0.17-0.71). The highest agreement was found on MRI (mean 0.56, range 0.20-0.77). The largest variations of COM were found in anterior-posterior direction for all imaging modalities. CONCLUSIONS: Large inter-observer variation in GTV delineation exists for CT, CT-MRI and MRI. MRI-based GTV delineation resulted in larger volumes and highest consistency between observers.

Linkage disequilibrium for two X-linked genes in Sardinia and its bearing on the statistical mapping of the human X chromosome.

The distribution of four X-linked mutants (G6PD, Deutan, Protan and Xg) among lowland and once highly malarial populations of Sardinia discloses a clear-cut example of linkage disequiligrium between two of them (G6PD and Protan). In the same populations the distribution of G6PD-deficiency versus colorblindness of the Deutan type and the Xg blood-group is not significantly different from that expected at equilibrium. These data suggest indirectly that the loci for G6PD and Protan may be nearer to one another than those for G6PD and Deutan.

A comprehensive evaluation of treatment accuracy, including end-to-end tests and clinical data, applied to intracranial stereotactic radiotherapy.

BACKGROUND AND PURPOSE: A methodology is presented to quantify the uncertainty associated with linear accelerator-based frameless intracranial stereotactic radiotherapy (SRT) combining end-to-end phantom tests and clinical data. METHODS AND MATERIALS: The following steps of the SRT chain were analysed: planning computed tomography (CT) and magnetic resonance (MR) scans registration, target volume delineation, CT and cone beam CT (CBCT) registration and intrafraction-patient displacement. The overall accuracy was established with an end-to-end test. The measured uncertainties were combined, deriving the total systematic (ΣT) and random (σT) error components, to estimate the GTV-PTV margin. RESULTS: The uncertainty in the MR-CT registration was on average 0.40mm (averaged over AP, CC and LR directions). Rotational variations were smaller than 0.5° in all directions. Interobser variation in GTV delineation was on average 0.29mm. The uncertainty in the CBCT-CT registration was on average 0.15mm. Again, rotational variations were smaller than 0.5° in all directions. The systematic and random intrafraction displacement errors were on average 0.55mm and 0.45mm, respectively. The systematic and random positional errors from the end-to-end test were on average 0.49mm and 0.53mm, respectively. Combining these uncertainties resulted in an average ΣT=0.9mm and σT=0.7mm and an average GTV-PTV margin of 2.8mm. CONCLUSION: This comprehensive methodology including end-to-end tests enabled a GTV-PTV margin calculation considering all sources of uncertainties. This generic method can also be used for other treatment sites.

Lymphocytic infiltration of bronchioloalveolar adenomas in mice.

The proportion of mouse lung adenomas with associated lymphocytes was examined in relation to tumor histology, location within the lung, tumor invasiveness and surface lymphocyte markers. The highest proportion of lymphocyte positive tumors was found on the pleural surface in contact with lymphatics. Seventy eight percent of pleural Clara cell but only 16% of alveolar cell adenomas were positive for lymphocytes (P less than 0.001). The majority of tumor associated lymphocytes were Ig-positive, either IgG (53%) or IgA (45%). No correlation was found between lymphocytic infiltration and tumor invasiveness. Our data suggest that the affinity of lymphocytes for Clara cell adenomas may be determined by intrinsic differences between the two tumors rather than to biologic behavior.

BNCT microdosimetry at the tapiro reactor thermal column.

A thermal column is available for dosimetric and radiobiological studies by the fast reactor TAPIRO, located at the ENEA research centre Casaccia. The TAPIRO neutron field has been studied (in the frame of LNL BNCT project) with a tissue-equivalent proportional counter, which has worked alternatively with an ordinary tissue-equivalent cathode and with a boron-enriched cathode. Measurements have been performed with polyethylene caps of different thickness. Both the absorbed dose and the microdosimetric-calculated biological effective dose show a maximum at approximately 0.5 mg cm(-2) of depth. The different dose components have been calculated and the results are discussed.

Monte Carlo calculations of positron emitter yields in proton radiotherapy.

Positron emission tomography (PET) is a promising tool for monitoring the three-dimensional dose distribution in charged particle radiotherapy. PET imaging during or shortly after proton treatment is based on the detection of annihilation photons following the ß(+)-decay of radionuclides resulting from nuclear reactions in the irradiated tissue. Therapy monitoring is achieved by comparing the measured spatial distribution of irradiation-induced ß(+)-activity with the predicted distribution based on the treatment plan. The accuracy of the calculated distribution depends on the correctness of the computational models, implemented in the employed Monte Carlo (MC) codes that describe the interactions of the charged particle beam with matter and the production of radionuclides and secondary particles. However, no well-established theoretical models exist for predicting the nuclear interactions and so phenomenological models are typically used based on parameters derived from experimental data. Unfortunately, the experimental data presently available are insufficient to validate such phenomenological hadronic interaction models. Hence, a comparison among the models used by the different MC packages is desirable. In this work, starting from a common geometry, we compare the performances of MCNPX, GATE and PHITS MC codes in predicting the amount and spatial distribution of proton-induced activity, at therapeutic energies, to the already experimentally validated PET modelling based on the FLUKA MC code. In particular, we show how the amount of ß(+)-emitters produced in tissue-like media depends on the physics model and cross-sectional data used to describe the proton nuclear interactions, thus calling for future experimental campaigns aiming at supporting improvements of MC modelling for clinical application of PET monitoring.

Toxicity of chloroprocaine and sodium bisulfite on human neuroblastoma cells.

The effects of commercial and crystalline solutions of chloroprocaine (CP) (1.6 X 10(-3)-0.4 X 10(-3)M) and sodium bisulfite (SB) (0.8 X 10(-3)-0.08 X 10(-3)M) were studied on the multiplication of human neuroblastoma cells. These cells were chosen because of putative neurotoxicity associated with CP; cell multiplication (measured as colony-forming ability-CFA) was studied because CFA is a reliable in vitro test for drug toxicity at the cellular level. CFA was dramatically reduced (86%) after 20-hr exposure to commercial solutions of CP. Shorter exposure times (3 hr) resulted in a marginal toxic effect (32%). At similar concentrations and after 20-hr exposure time, CP crystalline solutions induced a 37.5% inhibition that decreased with decreased time in culture. Sodium bisulfite reduced cell multiplication to a degree that varied with different samples of SB. With a 3-hr exposure time, CFA was reduced 72-92% by SB-1 and 57-72% by SB-2. The variability of SB toxicity and the difference in toxicity with commercial and crystalline solutions of CP are discussed in terms of possible clinical toxicity.

Cell membrane fusion by chloroprocaine.

The cytotoxicity of the local anesthetics chloroprocaine, procaine, and lidocaine was studied in murine and human cells. Murine glial and hepatic cells, and human fibroblasts were individually exposed to chloroprocaine, procaine, and lidocaine in concentrations ranging from 1.6 X 10(-3) M to 0.2 X 10(-3) M. The cells of all three cell lines underwent membrane fusion after exposure to chloroprocaine as indicated by the presence of the high number of multinucleated cells in the cultures. The 0.8 X 10(-3) M concentration was the most fusogenic, and caused multinucleation in 30% of glial and hepatic cells, and in 23% of fibroblasts. Membrane fusion and multinucleation also occurred in mixed human and murine cell cultures that were exposed to 0.8 X 10(-3) M concentration of both commercial and crystalline solutions of chloroprocaine, with a portion of multinucleated cells that was 27 and 17%, respectively. Cell membrane fusion was not caused by procaine, lidocaine, sodium bisulfite (the antioxidant present in the commercial solutions of chloroprocaine), or chloro-aminobenzoic acid and diethylamino-ethanol (the two chloroprocaine metabolites). The fusogenic effect of chloroprocaine on cell membrane has not been previously described for any local anesthetic.

In vitro induction of somatic cell hybridization by the local anesthetic chloroprocaine.

Chloroprocaine, an aminoester local anesthetic commonly used for epidural block, has been found to induce interspecies somatic cell hybrids in vitro. Mixed cultures of human amniocytes and mouse hepatoma cells, deficient in hypoxanthine phosphoribosyl transferase, were exposed to 1.6, 0.8, or 0.4 X 10(-3)M chloroprocaine for 3 hr at 37 degrees C, then maintained for 3 weeks in a double-selective medium of hypoxanthine, aminopterin, and thymidine (HAT) and ouabain to eliminate the unfused parental cells. Clones of actively multiplying cells appeared in cultures exposed to 1.6 and 0.8 X 10(-3)M chloroprocaine. Chromosome analysis confirmed they were hybrids. Cultures treated with 0.8 X 10(-3)M chloroprocaine exhibited the highest frequency of cell hybridization (8.8 X 10(-5). The hybrid clones bore the morphologic characteristics of both parents although their growth pattern closely resembled the mouse parent. Procaine, sodium bisulfite (the antioxidant present in the commercial solutions of chloroprocaine), and the two chloroprocaine metabolites, chloroaminobenzoic acid and diethylaminoethanol, were nonfusogenic. The hybridogenic effect of chloroprocaine has not been previously described with other local anesthetics.