Valproic acid radiosensitizes anaplastic thyroid cells through a decrease of the DNA damage repair capacity.

BACKGROUND: Anaplastic thyroid cancer (ATC) represents a rare lethal human malignancy with poor prognosis. Multimodality treatment, including radiotherapy, is recommended to improve local control and survival. Valproic acid (VA) is a clinically available histone deacetylase inhibitor with a well-documented side effect profile. In this study, we aim to investigate the combined effect of VA with photon irradiation in vitro. METHODS: Anaplastic thyroid cancer cells (8505c) were used to investigate the radiosensitizing effect of VA. RESULTS: VA sensitized cells to photon irradiation. VA increased radiation-induced apoptosis and radiation-induced DNA damage measured by γH2AX foci induction. Furthermore, VA prolonged γH2AX foci disappearance over time in irradiated cells and decreased the radiation-induced levels of mRNA of key DNA damage repair proteins of the homologous recombination (HR) and the nonhomologous end joining (NHEJ) pathways. CONCLUSIONS: VA at a clinically safe dose enhance the radiosensitivity of 8505c cells through an increase in radiation-induced apoptosis and a disruption in the molecular mechanism of HR and NHEJ DNA damage repair pathways.

Robotic versus laparoscopic surgery for rectal cancer: a comparative cost-effectiveness study.

BACKGROUND: The differences between the costs of robotic rectal resection and of the laparoscopic approach are still not well known. The aim of this study was to evaluate the cost-effectiveness of robotic versus laparoscopic surgery. METHODS: We conducted an observational, comparative, prospective, non-randomized study on patients having laparoscopic and robotic rectal resection between February 2014 and March 2018 at the Sanchinarro University Hospital, Madrid. Outcome parameters included surgical and post-operative costs, quality adjusted life years (QALY) and incremental cost per QALY gained or the incremental cost effectiveness ratio (ICER). The primary endpoint was to compare cost effectiveness in the robotic and laparoscopic surgery groups. A willingness-to-pay of 20,000€ and 30,000€ per QALY was used as a threshold to determine the most cost-effective treatment. RESULTS: A total of 81 RRR and 104 LRR were included. The mean operative costs were higher for RRR (4307.09€ versus 3834.58€; p = 0.04), although mean overall costs were similar (7272.03€ for RRR and 6968.63€ for the LLR; p = 0.44). Mean QALYs at 1 year for the RRR group (0.8482) was higher than that associated with LRR (0.6532) (p = 0.018). At a willingness-to-pay threshold of 20,000€ and 30,000€ there was a 95.54% and 97.18% probability, respectively, that RRR was more cost-effective than LRR. CONCLUSIONS: Our data regarding the cost-effectiveness of RRR versus LRR shows a benefit for RRR.

First case of complete full robotic surgical resection of leiomyosarcoma of the right renal vein.

BACKGROUND: Leiomyosarcomas present high postoperative morbidity and poor prognosis [1]. In the literature, only few cases of localized small leiomyosarcoma have been described [2, 3]. These cases might benefit from a minimally invasive approach. Robotic surgery has been claimed to have several advantages over laparoscopy such as enhanced vision and instruments movements which might make more feasible the execution of this type of surgery where partial renal resection is required. METHODS: A 53-year-old female with a medical history of myeloid leukemia and with chronic renal failure (creatinine: 2.6) was referred to our hospital for an incidental finding of right perirenal tumor of almost 3 cm compatible with leiomyosarcoma arising from the right renal vein. RESULTS: The operation was performed using a Da Vinci Robotic Surgical System model Si (Intuitive Surgical, Sunnyvale, CA, USA).Robotic ports were placed in a standard configuration for minimally invasive right nephrectomy. The dissection started with the partial mobilization of the right liver and Kocher maneuver. After the identification of the inferior vena cava the tumor was finally localized and dissected. Resection ended with a partial right vein resection and suture. Pathological final exam confirmed the diagnosis with margins free from tumor. CONCLUSIONS: In selected cases, robotic resection of leiomyosarcoma might be a safe and feasible procedure in experienced hands.

Robotic versus laparoscopic surgery for rectal cancer: a comparative study of clinical outcomes and costs.

PURPOSE: The costs involved in performing robotic surgery present a critical issue which has not been well addressed yet. The aims of this study are to compare the clinical outcomes and cost differences of robotic versus laparoscopic surgery in the treatment of rectal cancer and to conduct a literature review of the cost analysis. METHODS: This is an observational, comparative study whereby data were abstracted from a retrospective database of patients who underwent laparoscopic and robotic rectal resection from October 2010 to March 2017, at Sanchinarro University Hospital, Madrid. An independent company performed the financial analysis, and fixed costs were excluded. RESULTS: A total of 86 robotic and 112 laparoscopic rectal resections were included. The mean operative time was significantly lower in the laparoscopic approach (336 versus 283 min; p = 0.001). The main pre-operative data, overall morbidity, hospital stay and oncological outcomes were similar in both groups, except for the readmission rate (robotic: 5.8%, laparoscopic: 11.6%; p = 0.001). The mean operative costs were higher for robotic surgery (4285.16 versus 3506.11€; p = 0.04); however, the mean overall costs were similar (7279.31€ for robotic and 6879.8€ for the laparoscopic approach; p = 0.44). We found four studies reporting costs, three comparing robotic versus laparoscopy costs, with all of them reporting a higher overall cost for the robotic rectal resection. CONCLUSION: Robotic rectal resection has similar clinical outcomes to that of the conventional laparoscopic approach. Despite the higher operative costs of robotic rectal resection, overall mean costs were similar in our series.

Dynamics of ultra-thin polystyrene with and without a (artificial) dead layer studied by resonance enhanced dynamic light scattering.

Using non-invasive, marker-free resonance enhanced dynamic light scattering, the dynamics of capillary waves on ultrathin polystyrene films' coupling to the viscoelastic and mechanical properties have been studied. The dynamics of ultrathin polymer films is still debated. In particular the question of what influence either the solid substrate and/or the fluid-gas interface has on the dynamics and the mechanical properties of films of glass forming liquids as polymers is in the focus of the present research. As a consequence, e.g., viscosity close to interfaces and thus the average viscosity of very thin films are prone to change. This study is focused on atactic, non-entangled polystyrene thin films on the gold surface. A slow dynamic mode was observed with Vogel-Fulcher-Tammann temperature dependence, slowing down with decreasing film thickness. We tentatively attribute this relaxation mode to overdamped capillary waves because of its temperature dependence and the dispersion with a wave vector which was found. No signs of a more mobile layer at the air/polymer interface or of a "dead layer" at the solid/polymer interface were found. Therefore we investigated the influence of an artificially created dead layer on the capillary wave dynamics by introducing covalently bound polystyrene polymer brushes as anchors. The dynamics was slowed down to a degree more than expected from theoretical work on the increase of density close to the solid liquid interface-instead of a "dead layer" of 2 nm, the interaction seems to extend more than 10 nm into the polymer.

An organizational model to improve the robotic system among general surgeons.

INTRODUCTION: Robotic surgery has gained wide acceptance in recent years. However its development is slower and the lack of high level experience with this technique is an important limitation. This manuscript discusses some of the reasons of it and aims to describe the organizational system we have progressively established in our center in order to improve the development of Robotic program in our surgical area. METHODS: Some points may be required to improve the robotic program development in a general surgical department, including: a broad availability of robotic system in a surgical area; an ideal setting area with mainly oncological and hepato-biliary-pancreatic disease; the need of a mainly young team; a broad application of the robotic system in more general surgical fields; a high motivation on robotic use; a departmental and institutional economical effort. We have tried to achieve these goals before starting the robotic program in our department at October 2010. RESULTS: From October 2010 until November 2013 a total of 170 procedures have been performed, 92% of them for malignant diseases. Conversion rate and overall morbidity was 5% and 19%, respectively. CONCLUSIONS: The organizational model defined in our center is facilitating the constant and progressive development of the robotic program. A broad and flexible availability of the robotic system, a progressive increase of young surgeons joining this technology as well as the institutional and departmental economical effort are the points with which the robotic system may increase its development in a surgical department.

Robotic-assisted gastrectomy compared with open resection: a comparative study of clinical outcomes and cost-effectiveness analysis.

In the last decade, there have clearly been important changes in the surgical approach of gastric cancer treatment due to an increased interest in the minimally invasive surgical approach (MIS). The higher cost of robotic surgery procedures remains an important issue of debate. The objective of the study is to compare the main operative and clinical outcomes and to assess the incremental cost-effectiveness ratios (ICERs) of the two techniques. This is a prospective cost-effectiveness and clinical study when comparing the robotic gastrectomy (RG) technique with open gastrectomy (OG) in gastric cancer. Outcome parameters included surgical and post-operative costs, quality-adjusted life years (QALY) and incremental cost per QALY gained or the incremental cost-effectiveness ratio (ICER). The incremental utility was 0.038 QALYs and the estimated ICER for patients was dominated by robotic approach. The probability that the robotic approach was cost effective was 94.04% and 94.20%, respectively, at a WTP threshold of 20,000€ and 30,000€ per QALY gained. RG for gastric cancer represents a cost-effective procedure compared with the standard OG.

Extrapulmonary gas exchange enhances brain oxygen in pigeons.

Blood in mouth, nose, and eye tissues of birds cools by evaporation, then flows to a cephalic vascular heat exchanger, the ophthalmic rete. There, acting as a heat sink, blood from the evaporative surfaces cools arterial blood flowing counter-current to it toward the brain. The brain thus remains cooler than the body core. Data for unanesthetized domestic pigeons (Columba livia) suggest that in addition to losing heat, blood perfusing the evaporative surfaces also exchanges oxygen and carbon dioxide with air. In the heat exchanger, this blood apparently gives up oxygen to, and gains carbon dioxide from, arterial blood. The consequent increase in oxygen and decrease in carbon dioxide in the brain's arterial blood enhance diffusion of these gases in, and oxygen supply to, the brain. Such events may help birds maintain the brain's oxygen supply during the high systemic demand of exercise and at the reduced oxygen availability of high altitude.

Functionalization of gum arabic including glycoprotein and polysaccharides for the removal of boron.

Bio hybrid material supported multifunctional (hydroxyproline with enriched glycoprotein) has been shown to be efficient in chelation with boron and can be used for removal of boron at ppm levels. Gum arabic (GUM) is biodegradable and nontoxic biopolymer. GUM includes GA-GP-GA-glycoprotein and polysaccharides. Hollow silica spheres (HSS) gain increasing attention, thanks to low density, high specific surface and good adsorption performance. In this study, sol-gel process was used for the preparation of HSS and was modified with epoxy group. The resulted epoxidized HSS (HSEPC) was reacted with GUM to obtain a poly saccharide derivative functional hollow silica sphere (HSGUM) for removal of boron. Characterizations of HSGUM were performed by FT-IR and SEM techniques. Adsorption isotherm and kinetic models were also applied to adsorption of boron onto HSGUM. The resulting sorbent HSGUM has been demonstrated efficient and the maximum boron sorption capacity at monolayer coverage (qmax) was found as average 4.10 mmol g-1.

Design and bioevaluation of a 32P-patch for brachytherapy of skin diseases.

The purpose of this study was to design and evaluate a 32P patch for brachytherapy of skin diseases. We employed Phosphoric-32P-acid and Chromic 32P-phosphate in combination with natural rubber or silicone to produce the patches. Stability studies in vitro to evaluate the leakage of radioactivity, autoradiographic studies to evaluate homogeneity and shielding, as well as therapeutic efficacy in an animal model of skin cancer of the selected 32P patch were performed. The 32P-silicone-patch demonstrated its safety for external application. Tumor growth was arrest and complete regressions of tumors were seen in some other cases with 40 Gy applied in a single-dose scheme. In conclusion, the 32P-silicone-patch is easy to prepare and use in the treatment of skin diseases.

Total pancreatoduodenectomy en bloc with superior mesenteric artery and vein resection after gemcitabine and nab-paclitaxel neoadjuvancy.

BACKGROUND: Pancreatectomy for locally advanced adenocarcinoma affecting the superior mesenteric artery (SMA) and superior mesenteric vein (SMV) is still under discussion [1]. However, in selected cases, in light of the advancement of recent neoadjuvant treatments, it must be taken into account [2,3]. This video demonstrates some of the technical aspects of SMA and SMV resection as well as some tips of vascular reconstruction. METHODS: A 48-year-old man with a large adenocarcinoma of the uncinated process affecting the SMA and SMV underwent 3 cycles of gemcitabine and nab-paclitaxel neoadjuvancy. Post chemotherapy studies showed no disease progression with a normalization of CA 19.9 and SUV of FDG PET CT scan and a downsizing of the tumor, as well. Therefore, an en bloc total spleno-pancreato-duodenectomy with resection of SMA and SMV was planned. RESULTS: Through a bilateral subcostal incision, an "arterial first approach" [3] was performed. Considering the large length of the vascular resection, the replacement of the resected SMA and SMV was performed using two PTFE grafts, as showed in the video. Postoperative pathology showed margins free from disease with an important pathological response (grade 2 of Ryan classification adapted from rectal cancer) [4]. The post-operative course was uneventful and the patient is still free from disease at 31 months from surgery. CONCLUSIONS: This case is part of a large experience our group have acquired since we started neoadjuvancy in 2010. In our experience, we gathered 25 cases of locally advanced pancreatic tumors, of which 12 underwent to pancreatic resection after good response to the neoadjuvant treatment. In 5 of them concomitant SMA and SMV resection was required and post-operative mortality occurred in 1 of them. Morbidities and mortalities are higher compared with standard pancreatectomies, specially related to the vascular reconstruction (bleeding, graft thrombosis) [5]. However, in some circumstances like young age, great radiological and biological response to neoadjuvancy (such as the case herein presented), surgery might be considered the best option of care providing the only possibility to increase survival for these types of locally advanced tumors. However, further studies are needed to know which patients might benefit from this approach. En bloc total spleno-pancreato-duodenectomy with resection of SMA and SMV might be considered as an effective procedure in selected cases of pancreatic adenocarcinoma with good response to preoperative treatment.

Up-regulation of vascular endothelial growth factor/vascular permeability factor in mouse skin carcinogenesis correlates with malignant progression state and activated H-ras expression levels.

Angiogenesis is a crucial process for tumor growth and metastasis regulated by the balance of positive and negative factors. Vascular endothelial growth factor (VEGF/VPF) is a specific mitogen for endothelial cells that has been shown to be overexpressed in a variety of tumors and other inflammatory diseases. To analyze the implication of VEGF/VPF during tumorigenesis, we have studied its expression at different stages of tumor development using the mouse skin carcinogenesis model. VEGF/VPF mRNA was induced in skin in vivo after 12-O-tetradecanoylphorbol-13-acetate treatment. Constitutive up-regulation of VEGF/VPF at the mRNA and protein levels was also observed in premalignant papillomas and, at a higher level, in squamous carcinomas, suggesting a correlation between VEGF/VPF expression and tumor progression. A direct positive correlation between VEGF/VPF mRNA expression and the level of activated H-ras gene was found in a series of cell lines representing different stages of epidermal tumor development. Consequently, a clone of one of these cell lines, HaCa4, which has lost most of its v-ras expression, down-regulated VEGF mRNA expression concomitantly with its metastatic potential. Direct evidence of H-ras involvement in VEGF induction was obtained when an immortalized mouse keratinocyte cell line transduced with a retrovirus carrying v-H-ras showed highly increased VEGF/VPF mRNA levels. These data show that in mouse skin carcinogenesis, the VEGF/VPF angiogenic stimulus occurs early during premalignant papilloma development and further increases at later stages. Moreover, we demonstrate that increasing the activated H-ras dose, a phenomenon that takes place sequentially throughout mouse skin tumor development, may play an additional role by facilitating malignant in vivo progression through the modulation of VEGF/VPF-mediated angiogenesis.

Predictive prognostic value of local and distant recurrence of F-fluorodeoxyglucose positron emission tomography for pancreatic neuroendocrine tumors with reference to World Health Organization classifications (2004, 2010). Case series study.

BACKGROUND: F-flurodeoxyglucose positron emission tomography (FDG-PET) have been claimed to be an important prognostic tool in different malignancies. However, its predictive prognostic value on pancreatic neuroendocrine tumors (PNETs) is still under investigation. AIM: We study the prognostic impact of FDG-PET scan in neuroendocrine pancreatic tumors. METHODOLOGY: Between 2007 and 2012, 26 patients with no metastastatic histologically confirmed PNETs (mean age: 57 years) were examined with FDG-PET. We studied its captation in relation with the well established hystopathological prognostic markers assessed in the tumoral resected specimen according to the WHO 2004 and ENETS/WHO 2010 classification. RESULTS: FDG-PET captation was positive in 17 cases (65.4%). The median follow-up period was 34.4 months and recurrences occurred in 4 cases (15.4%). We found a significant correlation between this captation and Ki 67 index (p = 0.032), mitotic index (p = 0.002), tumor grade (p = 0.017) and tumor size (p = 0.01). CONCLUSIONS: FDG-PET provides a good prognostic value for PNETs. Present results must be further validated with larger sample studies.

Preoperative treatment with gemcitabine plus nab-paclitaxel is a safe and effective chemotherapy for pancreatic adenocarcinoma.

INTRODUCTION: Recently, novel chemotherapeutic agents like nab-paclitaxel and gemcitabine demonstrated a survival benefit over gemcitabine alone in metastatic pancreatic cancer. However, there are limited clinical results using this chemotherapy in potentially resectable pancreatic adenocarcinoma. Our aim is to report the oncological results of patients affected by potentially resectable pancreatic adenocarcinoma that underwent surgery after a combination of gemcitabine and nab-paclitaxel. METHODS: A total of 25 patients have been included. We evaluated: (1) Drug toxicity; (2) tumoral response (tumoral size at CT scan, SUV of FDG PET-CT scan and CA 19.9; (3) resection rate; (4) R0 resection rate and histopathological response and (5) survival and disease free survival. RESULTS: Overall treatment was well tolerated. Treatment resulted in a statistical decrease of CA19-9 (p = 0.019) tumoral size (p = 0.04) and SUV (p = 0.004). The resection rate was 68% (17/25 patients). All specimens were R0 and 13 of 17 specimens had major pathological regressions (complete and important response). Median survival and medial disease free survival of patients that underwent surgery was 21 months and 19 months, respectively at a mean follow up of 38.5 months. CONCLUSIONS: This data suggests that nab-paclitaxel and gemcitabine is a safe and effective neoadjuvant treatment for potentially resectable pancreatic adenocarcinoma. This promising data should be confirmed in larger, randomized studies.

Site-specific gap-misrepair mutagenesis by O4-ethylthymine.

The mutagenicity of the DNA base O-alkylation adduct, O4-ethylthymine, specifically incorporated into the plasmid vector pUC8 at the unique SalI and HincII recognition sites, was studied in vivo. Escherichia coli, Micrococcus luteus and AMV DNA polymerases catalyze the incorporation of O4-ethylTMP against template adenine and guanine residues, resulting in DNA sequence alteration during subsequent replication in the host E. coli K-12 strain JM83. The greatest mutation frequency was observed with error-prone AMV DNA polymerase. High levels of cognate restriction endonuclease-resistant mutant plasmid isolates were obtained by gap replication repair in the presence of O4-ethylTTP. The yields of mutant isolates were dependent upon the relative concentration of the competing pyrimidine deoxynucleoside triphosphates, TTP and dCTP, in the misreplication reaction. Repair of incorporated O4-ethylTMP of plasmid DNA by in vitro treatment with specific alkyltransferase, prior to transformation in the host, effectively increases the mutagenic efficiency of the adduct. The results obtained are consistent with the high miscoding potential O4-ethylthymine observed in in vitro studies and its ability to base-pair with noncomplementary guanine residues in DNA.

Kinetic and inhibition studies of cinnamoyl-CoA reductase 1 from Arabidopsis thaliana.

Cinnamoyl coenzyme A reductase (CCR, EC 1.2.1.44), one of the key enzymes in the biosynthesis of lignin monomers, catalyzes the NADPH-dependent reduction of cinnamoyl-CoA esters to their corresponding cinnamaldehydes. AtCCR1, one of the two distinct isoforms isolated from Arabidopsis thaliana, was shown to be involved in lignin biosynthesis during development. Here, we report on the purification of the recombinant AtCCR1 protein expressed in Escherichia coli and the subsequent determination of its kinetic properties (K(m) and k(cat)/K(m) values) towards its main substrates i.e. feruloyl-CoA, sinapoyl-CoA, and p-coumaroyl-CoA esters. In addition, the potential inhibitory effect of five substrate-like analogs possessing an N-acetylcysteamine thioester group was tested on CCR activity using either feruloyl-CoA or sinapoyl-CoA as substrates. The K(i) values were in the range of 4.4-502 microM and the type of inhibition was found to be either uncompetitive or noncompetitive. Interestingly, for compounds 3 and 5, the type of inhibition was found to be different depending on the substrate used to monitor the enzyme activity. The best inhibitors were those possessing the feruloyl (compound 3) and sinapoyl (compound 5) aromatic moiety (4.1 and 7.1 microM) while the enzyme activity was monitored using the corresponding substrates.

Is "small for size syndrome" a relatively new complication after the ALPPS procedure?

Aim of this study is to assess the incidence and outcome of small for size syndrome after ALPPS procedure. This novel procedure is claimed to induce accelerated and increased growth of future liver remnant after major hepatectomies. We prospectively collected data on nine consecutive patients undergoing ALPPS procedure at our center. Main patients' characteristics, outcome and postoperative complications, including small for size syndrome were analyzed. Main interval between two stages of ALPPS was 15.1 days. Mean future liver remnant hypertrophy was 93.2%. Severe complications occurred in 44.4% of patients and mortality occurred in two cases (22.2%). Small for size syndrome occurred in two cases (22.2%). The first one was detected intraoperatively during first stage. It was successfully managed by spleno-renal venous shunt. Second case was not promptly detected and died on postoperative day 64. This case series provides evidence that SFFS can be an important complication after ALPPS procedure. If it is not promptly detected and properly managed it can be a cause of death as occurred in our case.

Role of distinct subpopulations of peritoneal macrophages in the regulation of reactive oxygen species release.

It has been reported in vitro that during the respiratory burst of phagocytic cells the superoxide anion production per cell shows a negative relation with the cell density. This process has been described as autoregulation. The aim of this work was to analyze the superoxide anion production in thioglycollate-elicited peritoneal macrophage exudates to evaluate the importance of the peritoneal cavity environment in the autoregulation process. 12-O-tetradecanoylphorbol-13-acetate (PMA) was used to stimulate the respiratory burst and superoxide anion production was measured evaluating the intracellular formazan deposits that precipitate as a result of nitro blue tetrazolium (NBT) reduction. We have demonstrated a negative correlation between superoxide anion production and cell density in the peritoneal cavity in macrophages challenged with PMA. The response of individual cells was analyzed by means of an image analyzer, measuring the amount of formazan per cell and cell-size changes during the process of activation. The results revealed that the decrease in individual cell response as a function of higher cell densities were due to a significant increase in the amount of basal reaction macrophages. Concomitantly, the number of reactive cells remained unchanged irrespective of the cell density of the population. A direct correlation between cell size and superoxide anion production was observed. This phenomenon was demonstrated in SENCAR and Balb/c strains. However, macrophages from SENCAR mice showed greater superoxide anion production than those from Balb/c. The differences between strains could be associated to the increased sensitivity to PMA tumor promotion of SENCAR mice. Based on this property, macrophages from SENCAR mice were stimulated with opsonized zymosan, a particulate stimulus that reflects the interaction macrophage-microorganism during the phagocytic process. This data will contribute to the knowledge of infection control. We conclude that variations in basal reaction cells modulates the macrophage activation response when excess macrophages are recruited to the peritoneum. This is demonstrated using different stimuli, thus suggesting that this response may be applied to a wide variety of stimuli-macrophage interactions. The differences between strains may be associated to the increased sensitivity to PMA tumor promotion of SENCAR mice.

Inhibition of benzoyl peroxide-induced tumor promotion and progression by copper(II)(3,5-diisopropylsalicylate)2.

The ability of a biomimetic superoxide dismutase agent, copper(II)(3,5-diisopropylsalicylate)2 (CuDIPS), to modulate benzoyl peroxide (BzPo)-induced tumor promotion and progression in mouse skin multistage carcinogenesis was evaluated. The results showed a significant inhibition of tumor incidence by CuDIPS pretreatment during promotion-progression. Different types of tumors were developed: papillomas, keratoacanthomas and squamous cell carcinomas. There was a significant increase in the keratoacanthoma-papilloma ratio when the period of treatment with BzPo was prolonged, which was inhibited by CuDIPS pretreatment. CuDIPS induced a significant inhibition of malignant conversion. Our results suggest that reactive oxygen species could be important in BzPo-induced promotion and progression.

Nucleolar organizer regions as a marker of incipient transformation in a model of experimental carcinogenesis.

Nucleolar organizer regions stained selectively with a silver colloid technique (AgNOR) were evaluated during the process of tumour promotion in the skin of mice. Tumour promotion and control skin samples were processed for identification of AgNOR by light microscopy and submitted to a morphometric study of the following AgNOR-related variables: nuclear area (V.NUC); AgNOR number per nucleus (N.NOR); single AgNOR area (V.NOR); total AgNOR area per nucleus (TV.NOR) and proportion of nucleus occupied by AgNOR (TV.NOR/V.NUC). N.NOR exhibited significant differences between control and tumour tissue, but in the promotion period, N.NOR did not exhibit a significant rise until week 24. V.NOR and TV.NOR rose significantly as early as 2 weeks after the onset of promotion when the cells fail to exhibit unusual microscopic features. The significant increase in AgNOR material at the beginning of the promotion period reveals the potential value of the variables assessed in the early quantitative evaluation of cellular alterations which could be linked to the probability of tumour development. Rise in AgNOR material would indicate transcriptional activation leading to an increase in protein synthesis and, ultimately, to the expression of an altered phenotype.