NONO regulates the intra-S-phase checkpoint in response to UV radiation.

The main risk factor for skin cancer is ultraviolet (UV) exposure, which causes DNA damage. Cells respond to UV-induced DNA damage by activating the intra-S-phase checkpoint, which prevents replication fork collapse, late origin firing and stabilizes fragile sites. Recently, the 54-kDa multifunctional protein NONO was found to be involved in the non-homologous end-joining DNA repair process and in poly ADP-ribose polymerase 1 activation. Interestingly, NONO is mutated in several tumour types and emerged as a crucial factor underlying both melanoma development and progression. Therefore, we set out to evaluate whether NONO could be involved in the DNA-damage response to UV radiations. We generated NONO-silenced HeLa cell clones and found that lack of NONO decreased cell growth rate. Then, we challenged NONO-silenced cells with exposure to UV radiations and found that NONO-silenced cells, compared with control cells, continued to synthesize DNA, failed to block new origin firing and impaired CHK1S345 phosphorylation showing a defective checkpoint activation. Consistently, NONO is present at the sites of UV-induced DNA damage where it localizes to RAD9 foci. To position NONO in the DNA-damage response cascade, we analysed the loading onto chromatin of various intra-S-phase checkpoint mediators and found that NONO favours the loading of topoisomerase II-binding protein 1 acting upstream of the ATM and Rad3-related kinase activity. Strikingly, re-expression of NONO, through an sh-resistant mRNA, rescued CHK1S345 phosphorylation in NONO-silenced cells. Interestingly, NONO silencing affected cell response to UV radiations also in a melanoma cell line. Overall, our data uncover a new role for NONO in mediating the cellular response to UV-induced DNA damage.

New pyrazolo[3,4-d]pyrimidine SRC inhibitors induce apoptosis in mesothelioma cell lines through p27 nuclear stabilization.

Malignant mesothelioma (MM) is a highly aggressive tumor of the serous membranes for which there is currently no effective curative modality. Recent data suggest that hyperactivation of the tyrosine kinase SRC has a key role in MM development and therefore this kinase represents an important molecular target for MM therapy. We tested new pyrazolo[3,4-d]pyrimidine SRC inhibitors on a panel of MM cell lines expressing the active form of SRC. These SRC inhibitors exerted a significant proapoptotic effect on MM cells without affecting the normal mesothelial cell line MET-5A, supporting a possible use of these SRC inhibitors for a safe treatment of MM. We also showed that SRC inhibitor-induced apoptosis occurred concomitantly with an increase in the nuclear stability of the cyclin-dependent kinase inhibitor p27. This finding is remarkable considering that loss of nuclear p27 expression is a well-established adverse prognostic factor in MM, and p27 nuclear localization is crucial for its tumor-suppressive function. Consistently, SRC inhibition seems to promote the increase in p27 nuclear level also by inactivating the AKT kinase and downregulating cyclin D1, which would otherwise delay p27 nuclear import and provoke its cytoplasmic accumulation. To determine whether p27 stabilization has a direct role in apoptosis induced by SRC inhibition, we stably silenced the CDKN1B gene, encoding p27, in MSTO-211H and REN mesothelioma cells by transduction with lentiviral vectors expressing short hairpin RNAs against the CDKN1B transcript. Strikingly, p27 silencing was able to suppress the apoptosis induced by these SRC inhibitors in both MM cell lines, suggesting that p27 has a crucial proapoptotic role in MM cells treated with SRC inhibitors. Our findings reveal a new mechanism, dependent on p27 nuclear stabilization, by which SRC inhibition can induce apoptosis in MM cells and provide a new rationale for the use of SRC inhibitors in MM therapy.

Feline herpesvirus-1 down-regulates MHC class I expression in an homologous cell system.

Cytotoxic T lymphocytes (CTLs) are an essential component of the immune defense against many virus infections. CTLs recognize viral peptides in the context of the major histocompatibility complex (MHC) class I molecules on the surface of infected cells. Many viruses have evolved mechanisms to interfere with MHC class I expression as a means of evading the host immune response. In the present research we have studied the effect of in vitro Feline Herpesvirus 1 (FeHV-1) infection on MHC class I expression. The results of this study demonstrate that FeHV-1 down regulates surface expression of MHC class I molecules on infected cells, presumably to evade cytotoxic T-cell recognition and, perhaps, attenuate induction of immunity. Sensitivity to UV irradiation and insensitivity to a viral DNA synthesis inhibitor, like phosphonacetic acid, revealed that immediate early or early viral gene(s) are responsible. Use of the protein translation inhibitor cycloheximide confirmed that an early gene is primarily responsible.

A study on two different CAD systems for mammography as an aid to radiological diagnosis in the search of microcalcification clusters.

OBJECTIVE: The aim of the present study was to evaluate the efficacy of two different computer aided detection (CAD) systems for mammography in improving radiological diagnosis in the search of microcalcification clusters. The CAD systems used are: the SecondLooktrade mark (CADx Medical Systems, Canada) commercial system and the CALMA (computer assisted library in MAmmography) research CAD system. Three radiologists were asked to read mammographic images with and without the support of the CAD systems. MATERIAL AND METHODS: Three radiologists with respectively 3, 5 and 7 years of practice in mammogram reading in an Italian public hospital analysed a dataset composed of 120 digitized mammograms of healthy subjects with no lesion (proven by a radiological follow up of at least 3 years) and 70 images of patients with malignant cluster of microcalcification (proven by histopathological examination) both with no CAD support as well as with the help of the SecondLooktrade mark system. After 3 months they were asked to observe the same digitized mammograms with the assistance of the CALMA system. The radiologists worked independently and were unaware of the final diagnosis. The values of the area A(z) under the ROC curve, diagnostic sensitivity, specificity, positive and negative predictive values, and diagnostic accuracy were evaluated with and without the support of the CAD systems. The reading time and qualitative evaluations of each radiologist were also reported. RESULTS: With the support of the two CAD systems an improvement in A(z) area was obtained ranging from 0.01 to 0.04. Sensitivity increased from +8.6 to +15.7% and specificity decreased from 0.8 to 4.2%. CONCLUSION: In our study, not conditioned by the dataset, the CAD systems as second reader produced an increase in overall sensitivity of up to 15.7%, with a little decrease in specificity of up to 4.2%. Based on these results both CAD systems might be used in the current practise to improve the sensitivity values of conventional reading (radiologist alone). The results of this study show that no significant differences exist in term of A(z), sensitivity and specificity between CALMA and CADx.

A 700 MHz 1H-NMR study reveals apoptosis-like behavior in human K562 erythroleukemic cells exposed to a 50 Hz sinusoidal magnetic field.

PURPOSE: To study cell damage and possible apoptosis in K562 human erythroleukemic cells exposed for 2 h to an extremely low frequency (ELF) 50 Hz sinusoidal magnetic field with a magnetic induction of either 1 or 5 mT using high resolution proton nuclear magnetic resonance (1H-NMR) spectroscopy. MATERIALS AND METHODS: One-dimensional 1H-NMR spectra were obtained on whole K562 cells and perchloric acid extracts of these cells. In addition, two-dimensional 1H-NMR spectra were also acquired. Cell damage was examined by lactate dehydrogenase release and changes in cell growth were monitored by growth curve analyses, bromodeoxyuridine incorporation and Ki67 antigen localization. Cell death (necrosis and apoptosis) were also studied by using the chromatin dye Hoechst 33258. RESULTS: The variations in numerous metabolites observed with 1H-NMR reveal apoptosis-like behavior in response of K562 cells to ELF fields. CONCLUSION: 1H-NMR can be extremely useful in studying the effects of ELF fields on cells. In particular, the variations in metabolites which suggest apoptosis-like behavior occur when the cells are not identifiable as apoptotic by more traditional techniques.

Extremely low frequency (ELF) magnetic fields and apoptosis: a review.

In recent years, there has been increasing evidence that extremely low frequency magnetic fields might be linked to tumours, particularly with childhood leukaemia. In the same period, the role of apoptosis in the tumour process has also gained increasing importance. It is the purpose of this review to describe the apoptotic process, discuss selected papers in which apoptosis is examined in cells exposed to magnetic fields and describe the possible biophysical mechanisms responsible for changes in the apoptotic process in exposed cells. Despite some differences, as a whole, the literature seems to demonstrate that magnetic fields induce changes in apoptosis in cells exposed to different experimental protocols. In addition, the important role of ions, particularly of Ca2+, in the apoptotic process is also discussed, and one possible model for magnetic field action on apoptosis that brings together experimental observations of different nature is suggested and discussed.

Time-resolved contrast function and optical characterization of spatially varying absorptive inclusions at different depths in diffusing media.

The role of a spatially varying absorptive inhomogeneity located at different depths within a turbid material has been investigated. This inhomogeneity has been characterized by a spatially dependent Gaussian distribution of its absorption coefficient. The present study has been performed calculating the time-resolved contrast function in the framework of the first-order perturbative approach to the diffusion equation for a slab geometry and a coaxial measurement scheme. The model has allowed us to take into account different locations of the inclusion along the source-detector axis. The accuracy of time-resolved contrast predictions has been analyzed through comparisons with results of the finite element method that has been used to numerically solve the diffusion equation. Recovery of the absorption perturbation parameter of the inhomogeneity for different axial positions has also been investigated.

[Assessment of exposure to laser radiation in research laboratories]. / Valutazione dell'esposizione alla radiazione laser nei laboratori di ricerca.

The problem of laser safety is now topical due to the wide use of laser systems in different working environments. The program of prevention and protection prescribed by law 626/94 gives general rules for the elimination (where possible) and/or reduction of risk factors. For this purpose, it is important to determine the parameters that characterize such risks. The Maximum Permissible Exposure (MPE) and the Nominal Ocular Hazard Distance (NOHD) are the most important parameters of laser safety. The evaluation of these parameters requires a detailed knowledge of the standards and of the various techniques which are necessary to measure them. In particular, the MPE parameters are obtained by CEI EN 60825-1 standard in relation to wavelength and emission duration of the investigated laser. Exposure to laser radiation is usually measured in terms of irradiance (W/m2) or radiant exposure (J/m2). The experimental values of irradiance must be compared with the MPE parameters obtained by safety standards. When the values of irradiance exceed the MPE parameters then the NOHD values must to be calculated. The aim of this paper is to offer a general view of the methods for measuring the above-mentioned parameters for the laser sources that are most widely used in research environments and to compare such parameters with those recommended by safety standards. Our results indicate that for almost all the laser sources analysed, the measured exposure values were greater than the MPE recommended by standards, both for eyes and skin. High values of NOHD indicate that it is necessary to use eye protection, beam stops or attenuators at the end of the useful beam path in normal working conditions and especially in research laboratories in which these conditions can often change.

Fibronectin facilitates adhesion of K562 leukemic cells normally growing in suspension to cationic surfaces.

The role of protein adsorption in the forced adhesive growth of K562 leukemic cells onto a cationic surface composed of polylysine was investigated. Numerous studies have demonstrated that adhesion in anchorage-dependent cells is mediated in vitro by adsorption of serum proteins [particularly proteins of the extracellular matrix (ECM) such as fibronectin and vitronectin] present in the growth medium. Specifically, adhesion has been shown to occur when ECM proteins attach to the substratum and act as ligands for specific receptors located on the surface of cells. K562 cells are human erythroleukemic cells that normally grow in suspension. These cells are not involved in the same cell adhesion processes as anchorage-dependent cells and do not need to be attached to ECM proteins in order to survive and grow. Thus, with these systems, it is possible to better determine the role of protein adsorption in the adhesion of cells, growing in suspension such as blood cells, onto charged surfaces. The results presented show that adhesion of K562 cells onto the positively charged polylysine surface in the presence of serum is mediated through specific interactions between fibronectin receptors present on K562 cells and fibronectin adsorbed onto that cationic surface. Specifically, determination of cell adhesion under different experimental conditions indicates that nonspecific charge interactions do not take place directly between the cells and polylysine, but rather take place between polylysine and fibronectin, which adsorbs onto the cationic polymer. In addition, flow cytometric analyses reveal that only fibronectin receptors are present on these cells and, consequently, only fibronectin can be responsible for the actual adhesion of these cells onto the cationic surface. In view of the data presented, the possibility should be considered that ECM components adsorbed onto surfaces with specific charges and/or belonging to certain functional groups are involved in structural and functional modifications in cells. These cells grow in suspension and are normally not involved in adhesion phenomena, though these components should be considered. These considerations should be made especially when designing biomaterials that can modulate the response of cells growing in suspension, such as blood cells, and also in tissue engineering of blood substitutes.

Absence of in vivo DNA-protein interactions in the DQA2 and DQB2 promoter regions.

The DQ subregion of the human major histocompatibility complex (HLA) contains two pairs of loci: the DQA1/B1 genes (hereafter called DQ1), coding for the DQ molecules, and the DQA2/B2 pseudogenes (hereafter called DQ2). These pseudogenes are highly homologous to the functional DQ1 genes and they have no apparent abnormal features in their sequences that could prevent their activity. Only recently a low expression of the DQA2 gene has been observed whereas the DQB2 transcript was never found. The comparison between the DQ1 and DQ2 regulatory sequences revealed several differences in their W, X, and Y cis-acting elements. To examine the DNA/protein interactions in the DQ promoter regions, we performed in vivo footprinting experiments. Whereas the functional DQ1 loci showed a series of DNA-protein contact points in the X and Y boxes, the promoters of the DQ2 pseudogenes displayed an unoccupied phenotype. These findings suggest that the very low level of DQA2 expression and the apparent lack of DQB2 activity are caused by the reduced binding affinity of specific transcription factors.

Modulation of osteosarcoma cell growth and differentiation by silane-modified surfaces.

The effects of growing the Saos-2 human osteosarcoma cell line onto surfaces containing -CH(3), -OH, -COOH, -NH(2), and C6H5 groups obtained by silane modification were examined. These cells were used because of the great importance of bone cells in many aspects of biomaterials research. Silane-modified surfaces were characterized by contact angle measurements and, subsequently, surface energies were calculated. Cells grown on clean glass, as well as those grown on glass surfaces containing the functional groups cited above, were examined by light and scanning electron microscopy and assessed for their growth characteristics (i.e., determination of cell number and Ki67 antigen expression). The data presented seemed to indicate that if Saos-2 cells are grown on silane-modifed surfaces containing the methyl (CH(3)), hydroxyl (OH), and phenyl (C6H5) functional groups, their proliferation is slowed down while growth of these cells on glass surfaces modified with amino (NH(2)) and carboxyl (COOH) groups did not significantly affect growth. Once it was demonstrated that these three functional groups induce significant variations in proliferation, cells grown on these surfaces were also tested for apoptosis and expression of important markers of bone cell differentiation (i.e., osteonectin and osteopontin) by flow cytometry and eventual rearrangement of these markers by fluorescence microscopy. The data suggested that growth of Saos-2 cells on CH(3) induces the most evident morphological changes while growth of these cells on OH and C6H5 brings about the greater variations in osteonectin and osteopontin. We hypothesized that these changes are indicative of an increase in differentiation of Saos-2 cells when grown on the OH and C6H5 groups.

The relationship between 1H-NMR mobile lipid intensity and cholesterol in two human tumor multidrug resistant cell lines (MCF-7 and LoVo).

The high resolution proton nuclear magnetic resonance (1H-NMR) spectra of two different cell lines exhibiting multidrug resistance (MDR) as demonstrated by the expression of the well-known energy-driven, membrane-bound 170 kDa P-glycoprotein pump known as Pgp were investigated. In particular, the mobile lipid (ML) profile, and the growth and biochemical characteristics of MCF-7 (human mammary carcinoma) and LoVo (human colon adenocarcinoma) sensitive and resistant tumor cells were compared. The results indicate that both MCF-7 and LoVo resistant cells have a higher ML intensity than their respective sensitive counterparts. However, since sensitive and resistant cells of each pair grow in the same manner, variations in growth characteristics do not appear to be the cause of the ML changes as has been suggested by other authors in non-resistant tumor cells. In order to investigate further the origin of the ML changes, lipid analyses were conducted in sensitive and resistant cell types. The results of these experiments show that resistant cells of both cell types have a greater amount of esterified cholesterol and saturated cholesteryl ester and triglyceride fatty acid than their sensitive counterparts. From a thorough analysis of the data obtained in this paper utilizing numerous techniques including biological, biophysical and biochemical ones, it is hypothesized that cholesterol and triglyceride play a pivotal role in inducing changes in NMR ML signals. The importance of these lipid variations in MDR is discussed in view of the controversy regarding the origin of ML signals and the paramount role played by the Pgp pump in resistance.

Apoptosis, cell adhesion and the extracellular matrix in the three-dimensional growth of multicellular tumor spheroids.

In the last few years, it has become increasingly apparent that cell survival and death, especially apoptosis, strongly depend on cell adhesion and the extracellular matrix. In addition, it has also become clear that the use of three-dimensional multicellular tumor spheroids, which mimick more closely solid tumors in vivo, are a realistic experimental model to investigate many aspects of tumor biology. In the present review, after a general overview of the current knowledge regarding apoptosis, cell adhesion and the extracellular matrix, the results obtained utilizing multicellular tumor spheroids in these types of studies are discussed. The main conclusion that may be drawn from a synthesis of the literature on these topics is that investigations with multicellular tumor spheroids yield much useful information that is sometimes in contradiction to that obtained with monolayer cultures, but is closer to that derived from in vivo studies. Consequently, the authors encourage that these three-dimensional systems be used in many studies in which cell death and adhesion are being examined.

A time-domain algorithm for NMR spectral normalization.

Recently, a new method for quantitatively comparing NMR spectra of control and treated samples, in order to examine the possible occurring variations in cell metabolism and/or structure in response to numerous physical, chemical, and biological agents, was proposed. This method is based upon the utilization of the maximum superposition normalization algorithm (MaSNAl) operative in the frequency domain and based upon maximizing, by an opportune sign variable measure, the spectral region in which control and treated spectra are superimposed. Although the frequency-domain MaSNAl algorithm was very precise in normalizing spectra, it showed some limitations in relation to the signal-to-noise ratio and to the degree of diversity of the two spectra being analyzed. In particular, it can rarely be applied to spectra with a small number of visible signals not buried in the noise such as generally in vivo spectra. In this paper, a time-domain normalization algorithm is presented. Specifically, it consists in minimizing the rank of a Hankel matrix constructed with the difference of the two free induction decay signals. The algorithm, denoted MiRaNAl (minimum rank normalization algorithm), was tested by Monte Carlo simulations as well as experimentally by comparing two samples of known contents both with the new algorithms and with an older method using a standard. Finally, the algorithm was applied to real spectra of cell samples showing how it can be used to obtain qualitative and quantitative biological information.

Forced adhesive growth of K562 leukemic cells that normally grow in suspension induces variations in membrane lipids and energy metabolism: a proton NMR study.

The mechanisms responsible for the adhesion of cells onto a material's surface and the effects that that adhesion may have on cell structure and function are fundamental questions in biomaterials research. We recently demonstrated that the erythroleukemic cell line K562, which normally grows in suspension, can be induced to grow attached to a polylysine-coated solid surface in an anchorage-dependent manner. In this study, the effects of the growth of K562 cells onto polylysine were further investigated utilizing 500 MHz 1H-NMR spectroscopy. The NMR results showed that when K562 cells are grown attached to a positively-charged polylysine surface, there are alterations in lipids and energy metabolism. In particular, there was a 31% increase in phosphatidylcholine and a 15% decrease in each of its two precursors, glycerophosphatidylcholine and choline, as well as a 20% increase in CH2 lipids and a 7% decrease in CH3 lipids in treated cells compared to the controls. These results suggest that adhesive growth can induce strong variations in membrane structure, including the membrane fluidity of K562 cells. In addition, in cells attached to polylysine there was about a 10% decrease in creatine (together with phosphocreatine), a 20% increase in gamma-glutamate, a 15% increase in beta-glutamate, and a 24% decrease in lactate. This second set of results, which is closely related to energy metabolism, indicates that not only does adhesive growth induce changes in K562 cell membrane structure, but also in the utilization of energy in these cells. The data are discussed in view of the possible role played by surface charge in affecting cell structure and function in cells that come into direct contact with charged biopolymers.

A new algorithm for NMR spectral normalization.

There is increasing use of high-resolution NMR spectroscopy to examine variations in cell metabolism and/or structure in response to numerous physical, chemical, and biological agents. In these types of studies, in order to obtain relative quantitative information, a comparison between signal intensities of control samples and treated or exposed ones is often conducted. The methods thus far developed for this purpose are not directly related to the overall intrinsic properties of the samples, but rather to the addition of external substances of known concentrations or to indirect measurement of internal substances. In this paper, a new method for quantitatively comparing the spectra of cell samples is presented. It depends on a normalization algorithm which takes into consideration all cell metabolites present in the sample. In particular, the algorithm is based on maximizing, by an opportune sign variable measure, the spectral region in which the two spectra are superimposed. The algorithm was tested by Monte Carlo simulations as well as experimentally by comparing two samples of known contents with the new method and with an older method using a standard. At the end, the algorithm was applied to real spectra of cell samples to show how it could be used to obtain qualitative and quantitative biological information.

Multicellular tumour spheroids in radiation biology.

PURPOSE: Multicellular tumour spheroids are being used with increasing frequency in various aspects of tumour biology, including studies dealing with radiation biology. This review attempts to outline recent studies using these three-dimensional systems in radiation biology with particular reference made to papers testing radiotherapeutic protocols with spheroids. DEFINITIONS: Multicellular tumour spheroids are three-dimensional structures composed of cancer cells. They are formed from monolayer tumour cells when these are grown by various in vitro methods (e.g. liquid-overlay, spinner flask and gyratory rotation systems). Because of the cellular organization in spheroids, they have been often shown to recreate in vivo tumours much more closely than two-dimensional in vitro models. CONCLUSIONS: Because of their particular architectural characteristics, multicellular spheroids are demonstrated to be extremely useful in testing radiotherapeutic protocols, including dose rate and fractionation, radioimmunotherapy and the effects of combined treatments (e.g. radiation and anti-neoplastic drugs). Further studies should seek not only to continue testing these protocols, but also to investigate the more fundamental questions of radiation-induced apoptotic cell death, cell-cycle events, cell-cell interactions and cell adhesion phenomena.

Experimental tests of different solutions to the diffusion equation for optical characterization of scattering media by time-resolved transmittance.

A detailed investigation of the use of time-resolved trasmittance for the optical characterization of scattering media by use of different analytical solutions to the diffusion equation has been performed. A femtosecond Ti:sapphire laser working at 800 nm and a streak camera with a time resolution of a few picoseconds were employed. Different latex and Intralipid solutions as well as biological samples were investigated. Reduced scattering coefficients were evaluated, and good agreement with the Mie predictions was found. An estimation of the order of magnitude of the absorption coefficient was obtained for the low-absorbance samples examined. These studies confirm experimentally that time-resolved trasmittance can be employed usefully for evaluating s values of thick scattering samples when a proper theoretical description that takes into account realistic boundary conditions is used.

Different binding of NF-Y transcriptional factor to DQA1 promoter variants.

Polymorphism in the HLA-DQA1 promoter (QAP) sequences could influence the gene expression through a differential binding of transcriptional factors. Considering the main role played by the Y-box in the transcription, we focused on the QAP4 variants differing for a G vs A transition from the QAP Y-box consensus sequence. Electrophoretic Mobility Shift Assay using the two Y-box sequences was performed to determine whether this mutation could be reflected in an allele-specific binding of transcriptional factors. Indeed, the NF-Y specific band, recognised by supershift experiments, was clearly observed using the Y-box consensus probe but it was barely detectable with the QAP4 one. On the contrary, two other complexes were found to more strongly interact with QAP4 Y-box in comparison to the consensus sequence. The analysis of a selected panel of HLA homozygous lymphoblastoid cell lines by competitive RT-PCR and by Northern blotting revealed that the DQA1 *0401, *0501,*0601 alleles regulated by the QAP4 promoters were less expressed at the mRNA level than the DQA1* 0201 allele regulated by the QAP2.1 variant. In conclusion, these results show an evident reduction of NF-Y binding to the mutated QAP4 Y-box and a decreased mRNA accumulation of the DQA1 alleles regulated by these variants.

[Assessment of radiation dose to patients in hysterosalpingography]. / Valutazione della dose di radiazioni alla paziente nell'isterosalpingografia.

INTRODUCTION: The optimization principle applied to the patient radioprotection, recently introduced in Italian legislation, requires a careful analysis of the working procedures and clinical protocols generally used in radiological practice, to avoid unnecessary exposures with no loss in diagnostic information. We carried out this analysis in hysterosalpingography, which is the radiological examination usually performed on fertile women to detect uterine and tubal conditions. MATERIALS AND METHODS: The dosimetric survey was carried out on 35 patients, 22 to 40 years old, to evaluate: a) entrance dose and dose x area product; b) doses to the most irradiated organs, that is ovaries and uterus; c) effective dose equivalent and effective dose. The doses were measured with LiF thermoluminescent dosimeters, while an ionization chamber enabled us to the calculate dose area product. Ovaries and uterus doses were calculated with a Monte Carlo program using skin entrance exposure data and some technical parameters of the examination. RESULTS: The good agreement of the results obtained with both dosimetric methods demonstrated their equivalence in this specific case where the use of a clinical protocol, with no lateral projection, made the dosimeters lie always within the X-ray beam. DISCUSSION AND CONCLUSIONS: The mean dose was 4.5 mGy for the ovaries and 6.2 mGy for the uterus. Following the recommendations of the International Commission on Radiological Protection, both effective dose equivalent, with a mean value of 2.0 mSv, and effective dose, with a mean value of 1.95 mSv, were estimated. In conclusion, our results show that the patient dose can be reduced by limiting the use of fluoroscopy, using small size films and decreasing the number of exposures.