Mercedes Villalobos

Breast cancer risk and the combined effect of environmental estrogens.

AbstractObjective: The present study aimed to determine whether the combined effects of environmental estrogens measured as the total effective xenoestrogen burden (TEXB-alpha) are a risk factor for breast cancer over and above the risk potentially linked to specific pesticides. Methods: We measured the levels of 16 organochlorine pesticides as well as TEXB in adipose tissue of 198 women at the time of breast cancer diagnosis. These were compared with findings in 260 age and hospital matched control women without breast cancer. Results: The median levels of p,p′-DDE (1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene), aldrin, endosulfan ether and lindane (the pesticides detected in >40% of the study population) were higher in cases than controls, although the differences did not reach statistical significance. After adjusting for potential confounders, the odds ratio (OR) for breast cancer in women with detectable levels of aldrin was 1.55 (95% confidence interval (CI) 1.00–2.40). Among the postmenopausal women, the OR for aldrin and lindane was 1.84 (95% CI 1.06–3.18) and 1.76 (95% CI 1.04–2.98), respectively. Among cases with body mass index (BMI) below the median (28.6 kg/m2), the OR was 3.42 (95% CI 1.22–9.58) for women in the highest quartile of TEXB-alpha versus those in the lowest. The subgroup of leaner postmenopausal women showed an increased risk (OR: 5.67; 95% CI 1.59–20.21) for those in the highest tertile versus those in the lowest. Conclusions: We found an increased risk for breast cancer in the leaner women, especially in the leaner postmenopausal subgroup, related to the TEXB-alpha. The pesticides aldrin and lindane are also individually associated with risk.

Relationship between p53 status and radiosensitivity in human tumour cell lines

We examined the relationship between p53 levels before and after irradiation, radiation-induced cell cycle delays, apoptotic cell death and radiosensitivity in a panel of eight human tumour cell lines. The cell lines differed widely in their clonogenic survival after radiation, (surviving fraction at 2 Gy: SF2=0.18-0.82). Constitutive p53 protein levels varied from 2.2 +/- 0.4 to 6.3 +/- 0.3 optical density units (OD) per 10(6) cells. p53 after irradiation (6 Gy) also varied between the cell lines, ranging from no induction to a 1.6-fold increase in p53 levels 4 h after treatment. p53 function was also assessed by G1 cell cycle arrest after irradiation. The cellular response to radiation, measured as G0/G1 arrest, and the induction of apoptosis were in good agreement. However, a trace amount of DNA ladder formation was found in two cell lines lacking G1 arrest. Overall cellular radiosensitivity correlated well with the level of radiation-induced G1 arrest (correlation coefficient r=0.856; P=0.0067), with p53 constitutive levels (r=0.874, P=0.0046), and with p53 protein fold induction (r=-0.882, P=0.0038). Our data suggest that (1) the constitutive p53 level, (2) G1 arrest after irradiation, or (3) the p53 protein response to radiation may be good predictive tests for radiosensitivity in some cell types.

Assessing the Use of p16INK4a Promoter Gene Methylation in Serum for Detection of Bladder Cancer

Objective: This study was undertaken to investigate whether hypermethylation in p16 INK4a gene promoter could serve as plasma biomarker of bladder cancer. Methods and Patients: We examined the p16 INK4a status using methylation-specific PCR in 86 cancer patients and 49 controls (31 healthy people and 18 patients with benign urological diseases). Results: The p16 INK4a methylation was found in 22% of the serum samples and in 26% of the bladder cancer biopsies; one of them with carcinoma in situ. The presence of hypermethylated p16 INK4a in serum seems to be a product from tumour cells because a strong statistical association was found between both matched DNA signals (p < 0:0001). Using the control group, the presence of methylated p16 INK4a in the serum of individuals with suspicion of bladder cancer was found to be associated with the tumour presence (p ¼ 0:0009). Aberrant p16 INK4a methylation was also observed in one non-cancer patient, which is undergoing further assessment. Conclusions: According with our results, methylation of p16 INK4a promoter may be involved in the bladder cancer genesis and the presence of p16 INK4a methylated in serum of these patients could be useful in the cancer diagnosis with values of sensitivity, specificity and positive predictive value of 0.226, 0.950 and 0.98, respectively. These figures support the use of methylated p16 INK4a as a new class of tumour marker in bladder cancer.

Radiation-induced DNA double-strand break rejoining in human tumour cells

Five established human breast cancer cell lines and one established human bladder cancer cell line of varying radiosensitivity have been used to determine whether the rejoining of DNA double-strand breaks (dsbs) shows a correlation with radiosensitivity. The kinetics of dsb rejoining was biphasic and both components proceeded exponentially with time. The half-time (t1/2) of rejoining ranged from 18.0 +/- 1.4 to 36.4 +/- 3.2 min (fast rejoining process) and from 1.5 +/- 0.2 to 5.1 +/- 0.2 h (slow rejoining process). We found a statistically significant relationship between the survival fraction at 2 Gy (SF2) and the t1/2 of the fast rejoining component (r = 0.949, P = 0.0039). Our results suggest that cell lines which show rapid rejoining are more radioresistant. These results support the view that, as well as the level of damage induction that we have reported previously, the repair process is a major determinant of cellular radiosensitivity. It is possible that the differences found in DNA dsb rejoining and the differences in DNA dsb induction are related by a common mechanism, e.g. conformation of chromatin in the cell.

Initial radiation-induced DNA damage in human tumour cell lines: a correlation with intrinsic cellular radiosensitivity.

The role of the initial DNA double-strand breaks (dsb) as a determinant of cellular radiosensitivity was studied in human breast and bladder cancer cell lines. Cell survival was measured by monolayer colony-forming assay as appropriate and differences in radiosensitivity were seen (alpha-values ranged from 0.12 to 0.54). After pulsed-field gel electrophoresis (PFGE) the initial slopes of dose-response curves were biphasic with a flattening of the curves above 30 Gy. When the frequency of DNA dsb induction was assessed using a mathematical model based on the DNA fragment size distribution into the gel lane, we found a statistically significant relationship between the number of DNA dsb induced and the corresponding alpha-values and fraction surviving after 2Gy (P = 0.0049 and P = 0.0031 respectively). These results support the view that initial damage is a major determinant of cell radiosensitivity.

Early and late skin reactions to radiotherapy for breast cancer and their correlation with radiation-induced DNA damage in lymphocytes

IntroductionRadiotherapy outcomes might be further improved by a greater understanding of the individual variations in normal tissue reactions that determine tolerance. Most published studies on radiation toxicity have been performed retrospectively. Our prospective study was launched in 1996 to measure the in vitro radiosensitivity of peripheral blood lymphocytes before treatment with radical radiotherapy in patients with breast cancer, and to assess the early and the late radiation skin side effects in the same group of patients. We prospectively recruited consecutive breast cancer patients receiving radiation therapy after breast surgery. To evaluate whether early and late side effects of radiotherapy can be predicted by the assay, a study was conducted of the association between the results of in vitro radiosensitivity tests and acute and late adverse radiation effects.MethodsIntrinsic molecular radiosensitivity was measured by using an initial radiation-induced DNA damage assay on lymphocytes obtained from breast cancer patients before radiotherapy. Acute reactions were assessed in 108 of these patients on the last treatment day. Late morbidity was assessed after 7 years of follow-up in some of these patients. The Radiation Therapy Oncology Group (RTOG) morbidity score system was used for both assessments.ResultsRadiosensitivity values obtained using the in vitro test showed no relation with the acute or late adverse skin reactions observed. There was no evidence of a relation between acute and late normal tissue reactions assessed in the same patients. A positive relation was found between the treatment volume and both early and late side effects.ConclusionAfter radiation treatment, a number of cells containing major changes can have a long survival and disappear very slowly, becoming a chronic focus of immunological system stimulation. This stimulation can produce, in a stochastic manner, late radiation-related adverse effects of varying severity. Further research is warranted to identify the major determinants of normal tissue radiation response to make it possible to individualize treatments and improve the outcome of radiotherapy in cancer patients.

Normalization of widespread pressure pain hypersensitivity after total hip replacement in patients with hip osteoarthritis is associated with clinical and functional improvements

OBJECTIVE To evaluate the relevance of ongoing nociceptive joint inputs to the maintenance of widespread pain hypersensitivity in patients with hip osteoarthritis (OA) and to determine whether a reversal in the widespread pressure hypersensitivity together with an improvement in pain and function occurs after total hip replacement in these patients. METHODS Forty patients with hip OA participated. Twenty patients underwent total hip replacement, and the other 20 patients were assigned to a waiting list. Pressure-pain thresholds (PPTs) over the second metacarpal bone and the gluteus medius, vastus medialis, vastus lateralis, and tibialis anterior muscles were assessed bilaterally with a pressure algometer before and 3 months after total hip replacement surgery. Assessments of pain intensity (by visual analog scale [VAS]), physical function (by the Western Ontario and McMaster Universities Osteoarthritis Index), and health status (by the Short Form 12 health survey and the EuroQol 5-domain index) were also performed. RESULTS Patients who underwent total hip arthroplasty exhibited a reduction in widespread pressure pain hyperalgesia (increases in PPTs) over local and distant pain-free areas, as compared with before surgery and as compared with the patients assigned to the waiting list. PPTs were related to hip pain intensity, and significant correlations were found between higher VAS scores and lower average PPTs over all points assessed (-0.409 < r < -0.306, P < 0.05). Patients who underwent total hip arthroplasty exhibited a greater decrease in pain intensity and greater increases in function and health status than did those who were on the waiting list. Changes in the intensity of hip pain were moderately associated with changes in pressure pain sensitivity in the hip arthroplasty group. CONCLUSION Normalization of widespread pressure pain hyperalgesia was found after successful hip joint replacement in patients with hip OA. Altered pain processing seems to be driven by ongoing peripheral joint pathology, which stresses the importance of reducing pain in OA.

Individualisation of radiotherapy in breast cancer patients: possible usefulness of a DNA damage assay to measure normal cell radiosensitivity

PURPOSE The purpose of this study was to determine whether the distribution of sensitivities in breast cancer patients, measured using a DNA damage assay on lymphocytes, is likely to provide sufficient discrimination to enable the reliable identification of patients with abnormal sensitivities. MATERIAL AND METHODS Radiosensitivity (x) was assessed in 226 samples of lymphocytes from unselected women with breast cancer and was quantified as the initial number of DNA double-strand breaks (dsb) induced per Gy and per DNA unit (200 Mbp). RESULTS The existence of an inter-individual variation in the parameter (x) is described through the range (0.40-4.72 dsb/Gy/DNA unit) of values found, which have been fitted to the mathematical model defined by the log-normal distribution (mu = 0.42+/-0.03; sigma = 0.52+/-0.03; R(2)=0.9475). A total of 189 patients received radiotherapy after surgical treatment. Among them, we have detected 15 patients who developed severe skin reactions and we have compared their radiosensitivity values with the rest of patients treated. CONCLUSIONS Our results suggest that DNA initial damage measured on lymphocytes offers an approach to predict the acute response of human normal tissues prior to radiotherapy. Values of x higher than 3.20 dsb/Gy/DNA unit theoretically should correspond to the highly radio-sensitive patients. Using the experimental results, we have calculated the strength of the test by means of the area under the receiver operator characteristic curves (A(Z)) to determine whether the radiosensitivity assay can discriminate between patients according to their radiation response. The value found (A(Z)=0.675+/-0.072) is indicative of a fair-poor discriminating capacity of the test to identify the patients with higher risk of developing a severe acute reaction during the radiotherapy treatment.

DNA damage and prediction of radiation response in lymphocytes and epidermal skin human cells.

The success of radiotherapy in eradicating tumours depends on the total radiation dose, but what limits this dose is the tolerance of the normal tissues within the treatment volume. Studies involving fibroblast survival have demonstrated the theoretical feasibility of a predictive assay of radiation sensitivity, but such an assay is still far from clinical application. Using pulsed‐field gel electrophoresis (PFGE), we have quantified the initial “apparent” number of DNA double‐strand breaks (dsb) induced by the radiation as an alternative measure of sensitivity in 2 different normal cell types from the same patients, epidermal skin cells and lymphocytes. We found significant inter‐individual variation in the measured dsb (1–5 dsb/Gy/DNA unit). We also found a linear correlation between molecular damage in lymphocytes and skin samples from the same patient (slope = 0.83; r = 0.694; p = 0.0001). These results suggest that the initial number of dsb could be used as an indicator of the in vivo response to radiation. Int. J. Cancer 76:354–361, 1998.© 1998 Wiley‐Liss, Inc.

Radiation-Induced DNA Damage as a Predictor of Long-Term Toxicity in Locally Advanced Breast Cancer Patients Treated with High-Dose Hyperfractionated Radical Radiotherapy

Abstract Pinar, B., Lara, P. C., Lloret, M., Bordón, E., Núñez, M. I., Villalobos, M., Guerrero, R., Luna, J. D. and Ruiz de Almodóvar, J. M. Radiation-Induced DNA Damage as a Predictor of Long-Term Toxicity in Locally Advanced Breast Cancer Patients Treated with High-Dose Hyperfractionated Radical Radiotherapy. Radiat. Res. 168, 415–422 (2007). This 14-year-long study makes a novel contribution to the debate on the relationship between the in vitro radiosensitivity of peripheral blood lymphocytes and normal tissue reactions after radiation therapy. The aims were (1) to prospectively assess the degree and time of onset of skin side effects in 40 prospectively recruited consecutive patients with locally advanced breast cancer treated with a hyperfractionated dose-escalation radiotherapy schedule and (2) to assess whether initial radiation-induced DNA damage in peripheral blood lymphocytes of these patients could be used to determine their likelihood of suffering severe late damage to normal tissue. Initial radiation-induced DNA double-strand breaks (DSBs) were assessed in peripheral blood lymphocytes of these patients by pulsed-field electrophoresis. Acute and late cutaneous and subcutaneous toxicity was evaluated using the Radiation Therapy Oncology Group morbidity score. A wide interindividual variation was observed in toxicity grades and in radiation-induced DNA DSBs in peripheral blood lymphocytes (mean 1.61 ± 0.76 DSBs/Gy per 200 MBp, range 0.63– 4.08), which were not correlated. Multivariate analysis showed a correlation (P < 0.008) between late toxicity and higher prescribed protocol dose (81.6 Gy). Analysis of the 29 patients referred to 81.6 Gy revealed significantly (P < 0.031) more frequent late subcutaneous toxicity in those with intrinsic sensitivity to radiation-induced DNA DSBs of >1.69 DSBs/Gy per DNA unit. Our demonstration of a relationship between the sensitivity of in vitro-irradiated peripheral blood lymphocytes and the risk of developing late toxic effects opens up the possibility of predicting normal tissue response to radiation in individual patients, at least in high-dose non-conventional radiation therapy regimens.