Identification of an AP1-ZFP36 Regulatory Network Associated with Breast Cancer Prognosis.

It has been established that ZFP36 (also known as Tristetraprolin or TTP) promotes mRNA degradation of proteins involved in inflammation, proliferation and tumor invasiveness. In mammary epithelial cells ZFP36 expression is induced by STAT5 activation during lactogenesis, while in breast cancer ZFP36 expression is associated with lower grade and better prognosis. Here, we show that the AP-1 transcription factor components, i.e. JUN, JUNB, FOS, FOSB, in addition to DUSP1, EGR1, NR4A1, IER2 and BTG2, behave as a conserved co-regulated group of genes whose expression is associated to ZFP36 in cancer cells. In fact, a significant down-modulation of this gene network is observed in breast, liver, lung, kidney, and thyroid carcinomas compared to their normal counterparts. In breast cancer, the normal-like and Luminal A, show the highest expression of the ZFP36 gene network among the other intrinsic subtypes and patients with low expression of these genes display poor prognosis. It is also proposed that AP-1 regulates ZFP36 expression through responsive elements detected in the promoter region of this gene. Culture assays show that AP-1 activity induces ZFP36 expression in mammary cells in response to prolactin (PRL) treatment thorough ERK1/2 activation. These results suggest that JUN, JUNB, FOS and FOSB are not only co-expressed, but would also play a relevant role in regulating ZFP36 expression in mammary epithelial cells.

Rhomboid family gene expression profiling in breast normal tissue and tumor samples.

Rhomboid is an evolutionary conserved and functionally diversified group of proteins composed of proteolytically active and inactive members that are involved in the modulation of multiple biological processes such as epidermal growth factor receptor signaling pathway, endoplasmic reticulum-associated degradation, cell death, and proliferation. Recently, several human rhomboid genes have been associated with the development of chronic myeloid leukemia and pituitary, colorectal, ovarian, and breast cancers. In this study, we evaluated the mRNA and protein expression profiles of rhomboid genes in cancer cell lines and breast tissue/tumor samples. In silico analysis of publicly available gene expression datasets showed that different rhomboid genes are specifically expressed according to the breast cancer intrinsic subtypes. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis showed a significant RHBDD2 mRNA overexpression in advanced breast cancer compared with normal tissue samples (p = 0.012). In addition, we found that RHBDL2 and PARL mRNA expression was associated with a low/intermediate histologic tumor grade (p = 0.024 and p = 0.015, respectively). Immunohistochemistry analysis showed a significant increase of RHBDD2 protein expression in association with breast cancer samples negative for progesterone receptor (p = 0.015). Moreover, protein expression analysis corroborated the quantitative RT-PCR results, indicating that breast primary tumors belonging to patients with a more disseminated disease expressed significantly increased levels of RHBDD2 protein compared with less disseminated tumors (p = 0.01).

RHBDD2: a 5-fluorouracil responsive gene overexpressed in the advanced stages of colorectal cancer.

In previous studies, we identified rhomboid domain containing 2 (RHBDD2) gene to be markedly overexpressed in breast cancer patients that developed recurrence of the disease. In this study, we evaluated for the first time RHBDD2 gene expression in colorectal cancer (CRC). Five public available DNA microarray studies were compiled in a homogeneous dataset of 906 colorectal samples. The statistical analysis of these data showed a significant increase of RHBDD2 expression in the advanced stages of CRC (p < 0.01). We validated these findings by immunohistochemistry on 130 colorectal tissue samples; RHBDD2 protein overexpression was also observed in the advanced stages of the disease (p < 0.001). In addition, we investigated RHBDD2 expression in response to the chemotherapy agent 5-fluorouracile (5FU). We detected a significant increase of RHBDD2 mRNA and protein after 5FU treatment (20-40 µM; p < 0.001). Overall, these results showed that RHBDD2 overexpression might play a role in colorectal cancer progression.

Rhomboid domain containing 2 (RHBDD2): a novel cancer-related gene over-expressed in breast cancer.

In the course of breast cancer global gene expression studies, we identified an uncharacterized gene known as RHBDD2 (Rhomboid domain containing 2) to be markedly over-expressed in primary tumors from patients with recurrent disease. In this study, we identified RHBDD2 mRNA and protein expression significantly elevated in breast carcinomas compared with normal breast samples as analyzed by SAGE (n=46) and immunohistochemistry (n=213). Interestingly, specimens displaying RHBDD2 over-expression were predominantly advanced stage III breast carcinomas (p=0.001). Western-blot, RT-PCR and cDNA sequencing analyses allowed us to identify two RHBDD2 alternatively spliced mRNA isoforms expressed in breast cancer cell lines. We further investigated the occurrence and frequency of gene amplification and over-expression affecting RHBDD2 in 131 breast samples. RHBDD2 gene amplification was detected in 21% of 98 invasive breast carcinomas analyzed. However, no RHBDD2 amplification was detected in normal breast tissues (n=17) or breast benign lesions (n=16) (p=0.014). Interestingly, siRNA-mediated silencing of RHBDD2 expression results in a decrease of MCF7 breast cancer cells proliferation compared with the corresponding controls (p=0.001). In addition, analysis of publicly available gene expression data showed a strong association between high RHBDD2 expression and decreased overall survival (p=0.0023), relapse-free survival (p=0.0013), and metastasis-free interval (p=0.006) in patients with primary ER-negative breast carcinomas. In conclusion, our findings suggest that RHBDD2 over-expression behaves as an indicator of poor prognosis and may play a role facilitating breast cancer progression.

Comparative Analysis of the Biosimilar and Innovative G-CSF Modulated Pathways on Umbilical Cord Blood-Derived Mononuclear Cells.

Biosimilars of granulocyte colony-stimulating factor (G-CSF) have been routinely introduced into clinical practice. However, not functional genomics characterization has been performed yet in comparison with the innovator G-CSF. This study aimed to evaluate the transcriptomic changes in an in vitro model of umbilical cord blood cells (UBC) exposed to G-CSF for the identification of their modulated pathways. Umbilical cord blood cells-derived mononuclear cells (MNCs) were treated with biosimilar and innovator G-CSF for further gene expression profiling analysis using a microarray-based platform. Comparative analysis of biosimilar and innovator G-CSF gene expression signatures allowed us to identify the most commonly modulated pathways by both drugs. In brief, we observed predominantly upmodulation of transcripts related to PI3K-Akt, NF-kappaB, and tumor necrosis factor (TNF) signaling pathways as well as transcripts related to negative regulation of apoptotic process among others. In addition, hematopoietic colony-forming cell assays corroborate the G-CSF phenotypic effects over UBC-derived MNCs. In conclusion, our study suggests that G-CSF impacts UBC-derived cells through the modulation of several signaling pathways associated with cell survival, migration, and proliferation. The concordance observed between biosimilar and innovator G-CSF emphasizes their similarity in regards to their specificity and biological responses.

RHBDD2 overexpression promotes a chemoresistant and invasive phenotype to rectal cancer tumors via modulating UPR and focal adhesion genes.

The current standard of care for locally advanced rectal cancer (RC) is neoadjuvant radio-chemotherapy (NRC) with 5-fluorouracil (5Fu) as the main drug, followed by surgery and adjuvant chemotherapy. While a group of patients will achieve a pathological complete response, a significant percentage will not respond to the treatment. The Unfolding Protein Response (UPR) pathway is generally activated in tumors and results in resistance to radio-chemotherapy. We previously showed that RHBDD2 gene is overexpressed in the advanced stages of colorectal cancer (CRC) and that it could modulate the UPR pathway. Moreover, RHBDD2 expression is induced by 5Fu. In this study, we demonstrate that the overexpression of RHBDD2 in CACO2 cell line confers resistance to 5Fu, favors cell migration, adhesion and proliferation and has a profound impact on the expression of both, the UPR genes BiP, PERK and CHOP, and on the cell adhesion genes FAK and PXN. We also determined that RHBDD2 binds to BiP protein, the master UPR regulator. Finally, we confirmed that a high expression of RHBDD2 in RC tumors after NRC treatment is associated with the development of local or distant metastases. The collected evidence positions RHBDD2 as a promising prognostic biomarker to predict the response to neoadjuvant therapy in patients with RC.

Breast cancer cutaneous metastases are associated to uMUC1 and sialyl Lewis x and to highly malignant primary tumors.

Breast cancer spreading to different organs have been related to different molecules and mechanisms, but cutaneous metastasis remains unexplored. Increasing evidence showed that MUC1 and some of its carbohydrate associated antigens may be implicated in breast cancer metastasis. In this study we analyzed these tumor markers in order to identify breast cancer cutaneous metastatic profiles. A cohort of 26 primary tumors from breast cancer patients with cutaneous metastases were included; also, cutaneous and lymphatic node metastatic samples and primary tumors from breast cancer patients without metastases were analysed. Immunohistochemical (IHC) studies demonstrated that both underglycosylated MUC1 (uMUC1) and sialyl Lewis x (sLex) to be positively associated with cutaneous metastatic primary tumors (p < 0.05). Notably, a high percentage of tumors with cutaneous metastases were characterized as triple negative and Her2+ tumors (37.5 % and 29 %, respectively). Some discordant results were found between primary tumors and their matched cutaneous metastases. To determine if MUC1 variants may be carriers of carbohydrate antigens, subcellular fractions from a cutaneous metastatic lesion were obtained, immunoprecipitated and analyzed by Western blot. We found that the isolated uMUC1 with a molecular weight of>200 kDa was also the site for binding of anti-sLex MAb; in coincidence, a high correlation of positive IHC expression of both markers was observed. Our findings confirm that breast cancer cutaneous metastases were associated to highly malignant primary tumors and sustain the hypothesis that u-MUC1 and sLe x may drive breast cancer cutaneous metastases.

Spatiotemporal expression of Rhomboid domain containing 2 (Rhbdd2) during rat development.

Over the last few years rhomboid genes have gained interest because of its association with cancer and neurodegenerative diseases. In previous studies, we demonstrated that human RHBDD2 is over-expressed in the advanced stages of breast and colorectal cancers, suggesting a favorable role in cell proliferation. So far little is known about the expression of RHBDD2 in other tissues and other species, and because of similarities between cancer and embryonic cells, this study focused on the evaluation of Rhbdd2 expression in embryonic and adult rat tissues. By IHC and RT-PCR, Rhbdd2 was identified in early stages of most tissues analyzed, with high expression in brain, spinal cord, kidney and embryonic skin. In adult tissues, the expression remained elevated while salivary glands became positive. Furthermore, Rhbdd2 showed a high expression in the most proliferative stages of the rat mammary gland. Indeed, similar findings were observed in the mouse mammary epithelial cell line HC11, in which Rhbdd2 resides in the Golgi apparatus, and at different stages of mouse mammary gland development. Therefore, Rhbdd2 would be implicated in embryonic and adult tissue proliferation.

Association between activated K-ras and c-erbB-2 oncogenes with "high-risk" and "low-risk" human papilloma virus types in preinvasive cervical lesions.

Clinical and epidemiological data have linked cervical cancer to the Human Papilloma Virus (HPV) infection. However, the presence of HPV infection alone is not enough to cause tumorigenesis, suggesting a role for additional host-cell genetic factors. The aim of the present work was to study the association of K-ras and c-erbB-2 mutations in cervical tissue samples with different grades of dysplasia and infected with HPV-6 ("low-risk" type) or HPV-16 and HPV-18 ("high-risk" types). Negative HPV-DNA samples were used as controls. The detection of K-ras and c-erbB-2 activation were performed by Artificial Refractory Mutation System (ARMS)-PCR and semiquantitative PCR, respectively. Statistical analysis showed a highly significant difference in K-ras codon 12 mutation frequency between high-risk and low-risk HPV-infected samples (p<0.05). On the other hand, amplification of the c-erbB-2 oncogene appeared associated to tissue samples infected with HPV-6 (p<0.003). Cervical carcinoma appears to arise from a series of well-characterized progressive histological changes, but the genetic alterations necessary for cervical tumorigenesis are not yet clear. These results raise the possibility for a role of certain proto-oncogenes and their activation in cervical neoplasia.

HLA-DQA1 allele typing by nonisotopic PCR-LIS-SSCP.

In the present study we used a simple and reliable method for HLA-DQA1 allele typing based on the single-stranded conformation polymorphism (SSCP) properties of DNA molecules obtained by PCR. The technique consists of PCR amplification of a DNA fragment comprising the second exon of the HLA-DQA1 gene, amplicon denaturation using a low ionic strength solution (LIS), and electrophoresis on a small native polyacrylamide gel, followed by a rapid silver staining procedure. In order to validate the technique and to obtain the allele patterns for the DQA1 gene, 50 cervical samples were typed using this methodology and the commercial Amplitype HLA DQA1 Amplification and Typing kit. All the alleles detected with the kit were characterized by the LIS-SSCP approach. This procedure proved to be useful for population screening and typing of the DQA1 gene as well as for detecting new alleles or mutations in the donor-recipient molecular matching of HLA class II genes.

[Human papillomavirus genotype distribution in cervical infections among woman in La Plata, Argentina]. / Distribución de los genotipos del virus papiloma humano en infecciones cervicales en mujeres de La Plata, Argentina.

Genital infection with human papillomavirus (HPV) is one of the most common sexually transmitted viral diseases. High risk HPV are now considered the main etiologic agent of cancer of the uterine cervix and their high-grade precursor lesions. The aim of the present study was to investigate the endemic HPV-genotype spectrum in a population of women from the city of La Plata, Argentina. With this purpose, 718 cervical scrapes or biopsies corresponding to 152 normal samples (Pap I/II), 84 samples classified as atypical squamous cells of undetermined significance (ASCUS), 100 condyloma, 279 low-grade squamous intraepithelial lesions (LGSIL), 82 high-grade squamous intraepithelial lesions (HGSIL), and 21 squamous cell carcinomas (SCC) were studied. The detection of HPV-DNA was performed by nested polymerase chain reaction, using My 09/11 and Gp 05/06. The viral genotypes were analyzed by single-stranded conformation polymorphisms, employing low ionic strength solution (LIS-SSCP). The overall prevalence of HPV infection was 75% in the analyzed population, with a frequency of 46% for normal cervix, 69% for ASCUS, 86% for condyloma, 80% for LGSIL, 98% for HGSIL and 100% for SCC. The most prevalent viral types were HPV 16 (35%), followed by HPV 6/11 (27% each one), HPV 33 (6%) and HPV 18 (5%). HPV 16 was the most prevalent viral type among women with LGSIL, HGSIL and SCC, representing 33%, 50% and 67% of the genital infections, respectively. HPV 6 and 11 were the most frequent viral types among samples classified as Pap I/II, ASCUS and condyloma. Women between 21 and 30 year old showed the highest prevalence of HPV positivity, compraising the 32.2% of total infections.

[Detection and genotyping of human papillomavirus (HPV) using PCR-LIS-SSCP]. / Detección y genotipificación del papillomavirus humano (HPV) por PCR-LIS-SSCP.

This study describes a fast and simple method for human papillomavirus (HPV) typing based on the polymerase chain reaction (PCR) amplification of a portion of the viral genome and single strand conformation polymorphism using low ionic strength solutions (LIS-SSCP). PCR products were obtained using My09/My11 and Gp5/Gp6 primers in a nested reaction. The band patterns corresponded to the plasmid HPV clones from HPV-6, -11, -16, -18, -31, -33 and -34. The SSCP minigels were stained with SYBR-Green II. In order to determine diagnostic applicability, 100 cervical samples were studied comprising liquid cytology and paraffin embedded biopsies from patients showing squamous intraepithelial lesions (SILs). The SSCP patterns obtained from the clinical samples and the HPV clones were similar when the same type was present. Therefore, the methodology proved to be efficient and with high reproducibility for the detection and typing of HPV in clinical samples.

[Utilization of polymerase chain reaction for detection and genotyping of Helicobacter pylori in gastric biopsy using paraffin: a retrospective study]. / Utilización de la reacción en cadena de la polimerasa para la detección y genotipificación de Helicobacter pylori en biopsias gástricas incluídas en parafina: un estudio retrospectivo.

BACKGROUND: Helicobacter pylori infection is presumed to be the major causal agent of chronic active gastritis in humans. The persistent infection with this pathogen would be an important factor in the pathogenesis of peptic ulcer and also gastric cancer. METHODS: We investigated relationship between H. pylori characteristics in 42 patients with normal mucosa or gastritis with minor changes and 40 patients with mild and severe gastritis. Detection and typing of vacA and cagA genes were performed using a polymerase chain reaction method. RESULTS: The analysis of vacA prevalence and the type (S1 or S2) showed non-significant differences between the two groups studied (p > 0.05). However, cagA analysis showed highly significant differences between the groups classified as normal tissue-weak gastritis and mild-severe gastritis (p < 0.0001; OR = 8.4; CI = 3.1-22.8). CONCLUSIONS: cagA status is associated to the grade of gastritis, finding higher frequencies of H. pylori cagA+ in the moderate-severe gastritis group. These highly significant differences could make cagA status a genetic marker for disease progress.

Identification of signaling pathways modulated by RHBDD2 in breast cancer cells: a link to the unfolded protein response.

Rhomboid domain containing 2 (RHBDD2) was previously observed overexpressed and amplified in breast cancer samples. In order to identify biological pathways modulated by RHBDD2, gene expression profiles of RHBDD2 silenced breast cancer cells were analyzed using whole genome human microarray. Among the statistically significant overrepresented biological processes, we found protein metabolism­with the associated ontological terms folding , ubiquitination, and proteosomal degradation­cell death, cell cycle, and oxidative phosphorylation. In addition, we performed an in silico analysis searching for RHBDD2 co-expressed genes in several human tissues. Interestingly, the functional analysis of these genes showed similar results to those obtained with the microarray data, with negative regulation of protein metabolism and oxidative phosphorylation as the most enriched gene ontology terms. These data led us to hypothesize that RHBDD2 might be involved in endoplasmic reticulum (ER) stress response. Thus, we specifically analyzed the unfolding protein response (UPR) of the ER stress process. We used a lentivirus-based approach for stable silencing of RHBDD2 mRNA in the T47D breast cancer cell line, and we examined the transcriptional consequences on UPR genes as well as the phenotypic effects on migration and proliferation processes. By employing dithiothreitol as an UPR inducer, we observed that cells with silenced RHBDD2 showed increased expression of ATF6, IRE1, PERK, CRT, BiP, ATF4, and CHOP (p <0.01). We also observed that RHBDD2 silencing inhibited colony formation and decreased cell migration. Based on these studies, we hypothesize that RHBDD2 overexpression in breast cancer could represent an adaptive phenotype to the stressful tumor microenvironment by modulating the ER stress response.

Breast cancer biomarker discovery in the functional genomic age: a systematic review of 42 gene expression signatures.

In this review we provide a systematic analysis of transcriptomic signatures derived from 42 breast cancer gene expression studies, in an effort to identify the most relevant breast cancer biomarkers using a meta-analysis method. Meta-data revealed a set of 117 genes that were the most commonly affected ranging from 12% to 36% of overlap among breast cancer gene expression studies. Data mining analysis of transcripts and protein-protein interactions of these commonly modulated genes indicate three functional modules significantly affected among signatures, one module related with the response to steroid hormone stimulus, and two modules related to the cell cycle. Analysis of a publicly available gene expression data showed that the obtained meta-signature is capable of predicting overall survival (P < 0.0001) and relapse-free survival (P < 0.0001) in patients with early-stage breast carcinomas. In addition, the identified meta-signature improves breast cancer patient stratification independently of traditional prognostic factors in a multivariate Cox proportional-hazards analysis.

MUC1 oncogene amplification correlates with protein overexpression in invasive breast carcinoma cells.

The MUC1 gene is aberrantly overexpressed in approximately 90% of human breast cancers. Several studies have shown that MUC1 overexpression is due to transcriptional regulatory events. However, the importance of gene amplification as a mechanism leading to the increase of MUC1 expression in breast cancer has been poorly characterized. The aim of this study was to evaluate the role of MUC1 gene amplification and protein expression in human breast cancer development. By means of real-time quantitative polymerase chain reaction and immunohistochemical methods, 83 breast tissue samples were analyzed for MUC1 gene amplification and protein expression. This analysis showed MUC1 genomic amplification and a positive association with the histopathological group in 12% (1 out of 8) of benign lesions and 38% (23 out of 60) of primary invasive breast carcinoma samples (P = 0.004). Array-comparative genomic hybridization meta-analysis of 886 primary invasive breast carcinomas obtained from 22 studies showed MUC1 genomic gain in 43.7% (387 out of 886) of the samples. Moreover, we identified a highly statistical significant association between MUC1 gene amplification and MUC1 protein expression assessed by immunohistochemistry and Western blot test (P < 0.0001). In conclusion, this study demonstrated that MUC1 copy number increases from normal breast tissue to primary invasive breast carcinomas in correlation with MUC1 protein expression.

Analysis of adenocarcinoma of the colon and rectum: detection of human papillomavirus (HPV) DNA by polymerase chain reaction.

OBJECTIVE: The aim of the present work was to evaluate the presence of human papillomavirus genotypes in malignant and normal mucosa of the colon and rectum in order to determine if a relationship exists between HPV infection and colon neoplasms. MATERIALS AND METHODS: Thirty normal colon tissues and 54 sporadic adenocarcinomas were screened for HPV positivity using nested-PCR. Detection of viral types 6, 11, 16, 18, 33, 34 and 51 was performed by the LIS-SSCP (Low Ionic Strength-Single Strand Conformational Polymorphism) procedure. RESULTS: Significant differences in high risk HPV infection were found between normal samples and adenocarcinomas (P < 0.001). Among the cases, an inverse association between HPV infection and Dukes staging was also found (P = 0.020). Finally, there was no significant association between HPV and some classical clinicopathological features, although a gradient of infection form rectum to cecum was evident. CONCLUSION: The present study demonstrates that HPV may infect the glandular mucosa of the colon and suggests a possible association between HPV and colorectal cancer.

Association of human papillomavirus viral load with HPV16 and high-grade intraepithelial lesion.

The aim of the present study was to evaluate the relationship between viral type and copy number of human papillomavirus (HPV) with respect to the grade of cervical disease, and also to identify the existence of an HPV type-dependent viral load effect. DNA from 275 exocervical specimens, previously evaluated for histologic diagnosis, were evaluated for HPV presence, HPV type, and viral load. Viral load determination was performed using the low stringency PCR method (LS-PCR). Significant differences were found between the samples infected with HPV16 with respect to the samples infected with other 'high-risk' viral types (HPV -18, -31, -33 or -51) and 'low-risk' types (P < 0.05). However, highly significant differences were found between the viral loads observed in the high-grade squamous intraepithelial lesions group and normal epithelium (OR = 8.53) or the low grade ones (OR = 3.10). Moreover, a high viral load was detected in the condyloma acuminatum group compared to the normal epithelia samples (p< 0.05). This work confirms the genotype-specific association of viral load to the presence of HPV16. Also, a trend to higher viral loads could be seen in the more compromised cervical lesions. An unexpected level of viral particles appeared associated to the condylomas. This fact could be explained by a productive infection with high levels of viral replication.

c-myc gene amplification detected in preinvasive intraepithelial cervical lesions.

The aim of the present work was to evaluate the c-myc gene amplification process in cervical samples and to analyze the relationship between the activation of this proto-oncogene and the cytologic and/or histologic status. Thirty-four normal cervical samples and 105 abnormal cervical tissue scrapes, previously used for PAP or histopathologic diagnosis, were analyzed for c-myc gene amplification. Detection of c-myc gene amplification was performed using a polymerase chain reaction (PCR)-based method known as target arbitrarily primed-PCR (TAP-PCR). For c-myc amplification, significant differences were found between normal samples and samples presenting different grades of lesions (P<0.001). A significant difference between high-grade squamous intraepithelial lesions (HG-SIL) and the other stages of cervical disease was also found (P<0.05). This study demonstrated that c-myc copy number increases according to the histologic grade of the lesion. These results could indicate that oncogene amplification takes place in preinvasive stages of cervical disease and could cooperate not only in tumor progression but also in cell transformation.

Association between copper deficiency and DNA damage in cattle.

Cattle hypocuprosis is the second most widespread mineral deficiency affecting grazing cattle. The consequences of hypocuprosis include a failure of copper metalloenzymes, many of which form part of the antioxidant defence system. This work focuses on the association between copper (Cu) plasma concentration and DNA damage in Aberdeen Angus cattle. Two-hundred and ninety-nine heparinized blood samples from 2-year-old Aberdeen Angus cows were obtained from different farms located in the Salado River basin, Argentina. Plasma copper level analysis was carried out in whole samples, while cytogenetic analysis and single cell gel electrophoresis assay (comet assay) were carried out in 82 and 217 samples, respectively. Cytogenetic analysis showed a significant increase in the frequency of abnormal metaphases in moderate/severe hypocupremic groups (groups B and C) in relation to the normocupremic group (group A) (4.5 and 1.5 abnormal metaphases/100 cells, respectively, P < 0.01). The Spearman correlation test showed a negative association between cupremic values and the yield of chromosomal aberrations (r = -0.708, P < 0.0001). In the comet assay greater migration was observed in cells from the hypocupremic group, from a median of 54 in the severe hypocupremic group to 31 in the normocupremic group (P < 0.01). Accordingly, the Spearman correlation test showed a significant positive relationship between copper levels and cells without DNA migration and a significant negative relationship between copper levels and cells with a tiny tail (P < 0.0001 in both cases). The results obtained show that hypocupremia in cattle is associated with an increase in the frequency of chromosomal aberrations as well as in DNA migration as assessed by the comet assay. Whereas the comet assay could differentiate copper plasma level groups, chromosomal aberrations only detected differences between normal and hypocupremic animals. The increase of DNA damage found in hypocupremic animals could be explained by higher oxidative stress suffered by these animals.