Active Nucleolar Organizer Regions Research Articles

Breaks in the 45S rDNA Lead to Recombination-Mediated Loss of Repeats.

rDNA repeats constitute the most heavily transcribed region in the human genome. Tumors frequently display elevated levels of recombination in rDNA, indicating that the repeats are a liability to the genomic integrity of a cell. However, little is known about how cells deal with DNA double-stranded breaks in rDNA. Using selective endonucleases, we show that human cells are highly sensitive to breaks in 45S but not the 5S rDNA repeats. We find that homologous recombination inhibits repair of breaks in 45S rDNA, and this results in repeat loss. We identify the structural maintenance of chromosomes protein 5 (SMC5) as contributing to recombination-mediated repair of rDNA breaks. Together, our data demonstrate that SMC5-mediated recombination can lead to error-prone repair of 45S rDNA repeats, resulting in their loss and thereby reducing cellular viability.

Cytogenetic markers for the characterization of Capsicum annuum L. cultivars

Karyotypic studies with conventional staining have been unsuccessful due to the uniformity of Capsicum chromosomes. In this study, we found diagnostic chromosome characters that permit to characterize cultivars; this is the first cytological characterization of both rDNAs (18S and 5S) in a species of Capsicum using a genus-specific probe and the most exhaustive in C. annuum to date. The heterochromatic banding patterns enabled us to identify cultivars, and fluorescent in situ hybridization (FISH) showed one 5S rDNA locus largely conserved within the cultivars, whereas high variation in the number of 18S rDNA loci was observed. One of the most obvious differences is the presence of an additional active nucleolar organizer region in pair #12 and the dispersal of inactive 18S rDNA signals. These results indicate that fluorochrome banding together with silver impregnation and FISH procedures are very useful for the identification of chromosomes and the interpretation of chromosomal variation between cultivars. The functional role of this variation is still uncertain, but our results show that copy number variation of repetitive DNA during the course of evolution might provide an excellent experimental system for studying genome rearrangements accompanying functional divergence in domesticated C. annuum.

Structure of nucleoli in first-order spermatocytes of selected free-living animal species

Nucleoli are the product of the activity of nucleolar organizer regions (NOR) in certain chromosomes. Their main functions are the formation of ribosomal subunits from ribosomal protein molecules and the transcription of genes encoding rRNA. Nucleoli are present in the nuclei of nearly all eukaryotic cells because they contain housekeeping genes. The size and number of nucleoli gradually decrease during spermatogenesis. Some of the material originating in the nucleolus probably migrates to the cytoplasm and takes part in the formation of chromatoid bodies (CB). Nucleolus fragmentation and CB assembly take place at the same stage of spermatogenesis. CB are involved in the formation of the acrosome, the migration of mitochondria to the midpiece, and the formation of the sperm tail fibrous sheath. The aim of the study was to characterize the nucleoli in the early prophase of spermatogenesis in the wild boar and the roe deer. The roe deer cells have larger nucleoli and a larger cell nucleus than the wild boar cells. The area of the nucleolus as a percentage of the total area of the nucleus was larger as well. The coefficients of variation for all parameters were higher in the roe deer. In the wild boar cells the nucleoli were mainly regularly shaped. The size of the nucleolus and the nucleus of the spermatocyte is a species-specific trait associated with karyotype and the number of nucleolar organizer regions in a given species.

Cytogenetic analysis of Scinaxauratus and Scinaxeurydice (Anura, Hylidae) with emphasis on cytotaxonomy.

Scinax Wagler, 1830 is a species-rich genus of amphibians with relatively few detailed chromosomal reports. In this work, cytogenetic analyses of Scinax auratus (Wied-Neuwied, 1821) and Scinax eurydice (Bokermann, 1968) were carried out based on conventional (Giemsa staining, Ag-NOR and C-banding) and cytomolecular (base-specific fluorochrome staining and fluorescence in situ hybridization – FISH of ribosomal probes) techniques. Both species shared the same karyotype, location of active nucleolar organizer regions on pair 11 and GC-rich heterochromatin, as reported for most species in Scinax ruber clade. Interpopulation chromosomal variation was observed in Scinax eurydice, indicating the occurrence of cryptic species. The mapping of 18S ribosomal genes by FISH is reported for the first time in both species.

Chromosomal organization of the ribosomal RNA genes in the genus Chironomus (Diptera, Chironomidae).

Chromosomal localization of ribosomal RNA coding genes has been studied by using FISH (fluorescence in situ hybridization) in 21 species from the genus Chironomus Meigen, 1803. Analysis of the data has shown intra- and interspecific variation in number and location of 5.8S rDNA hybridization sites in 17 species from the subgenus Chironomus and 4 species from the subgenus Camptochironomus Kieffer, 1914. In the majority of studied species the location of rDNA sites coincided with the sites where active NORs (nucleolus organizer regions) were found. The number of hybridization sites in karyotypes of studied chironomids varied from 1 to 6. More than half of the species possessed only one NOR (12 out of 21). Two rDNA hybridization sites were found in karyotypes of five species, three – in two species, and five and six sites – in one species each. NORs were found in all chromosomal arms of species from the subgenus Chironomus with one of them always located on arm G. On the other hand, no hybridization sites were found on arm G in four studied species from the subgenus Camptochironomus. Two species from the subgenus Chironomus – Chironomus balatonicus Devai, Wuelker & Scholl, 1983 and Chironomus “annularius” sensu Strenzke, 1959 – showed intraspecific variability in the number of hybridization signals. Possible mechanisms of origin of variability in number and location of rRNA genes in the karyotypes of species from the genus Chironomus are discussed.

Chromosomal characteristics and distribution of rDNA sequences in the brook trout Salvelinus fontinalis (Mitchill, 1814).

Brook trout Salvelinus fontinalis (Mitchill, 1814) chromosomes have been analyzed using conventional and molecular cytogenetic techniques enabling characteristics and chromosomal location of heterochromatin, nucleolus organizer regions (NORs), ribosomal RNA-encoding genes and telomeric DNA sequences. The C-banding and chromosome digestion with the restriction endonucleases demonstrated distribution and heterogeneity of the heterochromatin in the brook trout genome. DNA sequences of the ribosomal RNA genes, namely the nucleolus-forming 28S (major) and non-nucleolus-forming 5S (minor) rDNAs, were physically mapped using fluorescence in situ hybridization (FISH) and primed in situ labelling. The minor rDNA locus was located on the subtelo-acrocentric chromosome pair No. 9, whereas the major rDNA loci were dispersed on 14 chromosome pairs, showing a considerable inter-individual variation in the number and location. The major and minor rDNA loci were located at different chromosomes. Multichromosomal location (3–6 sites) of the NORs was demonstrated by silver nitrate (AgNO3) impregnation. All Ag-positive i.e. active NORs corresponded to the GC-rich blocks of heterochromatin. FISH with telomeric probe showed the presence of the interstitial telomeric site (ITS) adjacent to the NOR/28S rDNA site on the chromosome 11. This ITS was presumably remnant of the chromosome rearrangement(s) leading to the genomic redistribution of the rDNA sequences. Comparative analysis of the cytogenetic data among several related salmonid species confirmed huge variation in the number and the chromosomal location of rRNA gene clusters in the Salvelinus genome.

Cytogenetic studies in six species of Scinax (Anura, Hylidae) clade Scinax ruber from northern and northeastern Brazil.

Scinax species are still underrepresented in cytogenetic studies, mainly with respect to populations from northeastern and northern Brazil. In this study, we provide new chromosomal information on Scinax boesemani, S. camposseabrai, S. garbei, S. pachycrus, S. trilineatus and S. x-signatus, all belonging to clade S. ruber. They were collected at two locations in the Caatinga biome (northeastern Brazil) and at one in the Amazon (northern Brazil) biomes. Chromosomes were analyzed by conventional staining, C-banding, Ag-NOR staining, and fluorochrome staining. All species shared a modal diploid value of 2n = 24 and fundamental arm number (FN) of 48. Moreover, both chromosomal size and morphology were similar to other species in this Scinaxclade. C-banding revealed centromeric heterochromatin in all species, along with terminal species-specific C-bands in some species. Active nucleolar organizer regions (Ag-NORs) were identified at 11q in most species, except for S. boesemani and S. garbei (Ag-NORs at interstitial region of 8q). Differing from most anurans, GC-rich regions were not restricted to NORs, but also coincident with some centromeric and terminal C-bands. These data contribute to the cytotaxonomy of Scinax by providing chromosomal markers and demonstrating the occurrence of microstructural rearrangements and inversions on chromosomal evolution of Scinax.

Cytogenetic description of Ancistrus abilhoai (Siluriformes: Loricariidae) from Iguaçu River basin, southern Brazil.

The Iguaçu River basin is a tributary to the upper Paraná River in southern Brazil, and is considered an important aquatic ecoregion that, although having few species of fish, 51-71% of these are apparently endemic. Ancistrus abilhoai is one of three recently described species for this basin and is currently considered endemic to the basin. In this study, we present the chromosomal structure of two populations of Ancistrus abilhoai one collected in the Iguaçu River, in Paraná State, and another collected in the Timbó River, a tributary of the Iguaçu River, in the State of Santa Catarina. Karyotype analyzes were performed in 11 specimens from the Iguaçu River (four females and seven males) and 12 specimens (all males) from Timbó River, revealing 2n = 48 chromosomes with a karyotype formula of 22m + 14sm + 6st + 6a in both populations. Analysis of active nucleolar organizer regions (Ag-NORs) and fluorescent in situ hybridization (FISH) with 18S rDNA probes revealed the submetacentric pair 13 bearing marks at terminal positions on the short arms. Considered as plesiomorphic chromosomal markers in Loricariidae, asynteny 18S and 5S rDNA, and small amounts of heterochromatin were observed. In this study, the first chromosomal data of A. abilhoai are presented with comments on karyotypic characteristics of the genus.

Age-Dependent Change in the Morphology of Nucleoli and Methylation of Genes of the Nucleolar Organizer Region in Japanese Quail (<I>Coturnix japonica</I>) Model (Temminck and Schlegel, 1849) (Galliformes: Aves)

Nucleoli are the product of the activity of nucleolar organizer regions (NOR) in certain chromosomes. Their main functions are the formation of ribosomal subunits from ribosomal protein molecules and the transcription of genes encoding rRNA. The aim of the study was to determine the shape of nucleoli and analyse methylation in the gene RN28S in the spermatocytes of male Japanese quail (Coturnixjaponica) in two age groups. Nucleoli were analysed in cells of the first meiotic prophase. Their number and shape were determined and they were classified as regular, irregular or defragmented. In the cells of the young birds no defragmented nucleoli were observed, with regular and irregular nucleoli accounting for 97% and 3%, respectively. In the cells of older birds no regular nucleoli were observed, while irregular and defragmented nucleoli accounted for 37% and 67%, respectively. MSP (methylation-specific PCR) showed that the gene RN28S is methylated in both 15-week-old and 52-week-old quails. In recent years an association has been established between nucleolus morphology and cellular ageing processes.

Genetic differentiation among distinct karyomorphs of the wolf fish Hoplias malabaricus species complex (Characiformes, Erythrinidae) and report of unusual hybridization with natural triploidy.

In this study, genetic differentiation between karyomorphs A (2n = 42) and D (2n = 39/40) of the wolf fish Hoplias malabaricus, which is comprised of several cryptic species that present a wide variety of diploid chromosome numbers and sex chromosome systems, resulting in the identification of seven distinct karyomorphs (A-G), was investigated using a combination of molecular and cytogenetic tools. Deep sequence divergences for both karyomorphs were observed and indicate a long period of reproductive isolation between karyomorphs A and D. Additionally, one individual with 61 chromosomes was identified, which, as far as is known, is the first case of natural triploidy resulting from the hybridization between these highly differentiated karyomorphs of H. malabaricus. Molecular and cytogenetic analyses revealed that this allotriploid specimen carries two sets of maternal chromosomes from karyomorph D (2n = 40) and one set of chromosomes from karyomorph A (n = 21). Moreover, ribosomal sites and active nucleolus organizer regions from both parental contributors were found in the triploid hybrid. Considering the significant genetic distances between karyomorphs A and D, one of the primary reasons for the lack of recurrent reports of hybridization in the H. malabaricus species complex may be due to post-zygotic barriers, such as hybrid sterility or unviability.

Cytogenetic and identification of the nucleolus organizer region in Heliconia bihai (L.) L.

The genus Heliconia is not much studied and the number of existing species in this genus is still uncertain. It is known that this number relies between 150 to 250 species. In Brazil, about 40 species are native and known by many different names. The objective of this paper was to characterize morphometrically and to identify the NOR (active nucleolus organizer regions) by Ag-NOR banding of chromosomes of Heliconia bihai (L) L. Root meristems were submitted to blocking treatment in an amiprofos-methyl (APM) solution, fixed in methanol-acetic acid solution for 24 hours, at least. The meristems were washed in distilled water and submitted to enzymatic digestion with pectinase enzyme. The slides were prepared by dissociation of the root meristem, dried in the air and also on hot plate at 50°C. Subsequently, some slides were submitted to 5% Giemsa stain for karyotype construction and to a solution of silver nitrate (AgNO3) 50% for Ag-NOR banding. The species H. bihai has 2n = 22 chromosomes, 4 pairs of submetacentric chromosomes and 7 pairs of metacentric chromosomes, and graded medium to short (3.96 to 0.67 μM), with the presence of active NOR in pairs 1 and 2 and interphase cells with 2 nucleoli. These are the features of a diploid species.

Reduction of the argyrophilic nucleolar organizing region associated protein synthesis with age in buccal epithelial cells of healthy individuals.

Nucleolus organizer regions (NORs) consist of the rRNA coding gene family (rDNA) in the cell nucleus. The argyrophilic proteins are selectively stained with silver nitrate and bind these regions. It was reported that NOR (rDNA) activity decreases in human lymphocytes, fibroblasts and bone marrow with age. However, to our knowledge there have not been any studies related to the NORs in oral epithelial cells of healthy individuals. Our aim is to detect any correlation between age and Total AgNOR area/Total nucleus area (TAA/TNA) values in buccal epithelial cells of healthy individuals. Oral epithelial cells from 50 healthy individuals (age range of 2-80 years old) were spread onto a clean glass slide, air dried and fixed. Then the AgNOR staining protocol was performed on these cells. TAA/TNA ratio and AgNOR dots were calculated using software. From each person 50 oral epithelial cells were evaluated. Statistically significant correlations were found between mean TAA/TNA values and age (Rsq = 0.534, p < 0.001 for linear and Rsq = 0.728, p < 0.0001 for polynominal regression), and between AgNOR number and age (Rsq = 0.621, p < 0.001 for linear and Rsq = 0.693, p < 0.0001 for polynominal regression). There is a significant correlation between age and AgNOR amount (ribosome biosynthesis rate) in buccal epithelial cells of healthy individuals. AgNORs in buccal epithelial cells may be used for detection of age.

Analysis of the proliferative potential of odontogenic epithelial cells of pericoronal follicles.

To evaluate the proliferative potential and the cell proliferation rate of odontogenic epithelial cells. Forty-two cases of pericoronal follicles of impacted third molars were submitted to silver impregnation technique for quantification of argyrophilic nucleolar organizer regions (AgNOR) and immunohistochemical staining for EGFR and Ki-67. For AgNOR quantification, the mean number of active nucleolar organizer regions per nucleus (mAgNOR) and the percentage of cells with 1, 2, 3 and 4 or more AgNORs per nucleus (pAgNOR) were quantified. Ki-67 immunolabeling was quantified, whereas for EGFR, a descriptive analysis of staining patterns (membrane, cytoplasm or membrane + cytoplasm positivity) was performed. We evaluated the reduced epithelium of the enamel organ and/or islands of odontogenic epithelium present in the entire connective tissue. mAgNOR were 1.43 (1.0-2.42) and were significantly different among pericoronary follicles from upper and lower teeth (p = 0.041). Immunostaining of Ki-67 was negative in all cases. EGFR immunolabeling was found mainly in the cytoplasm and was more intense in islands and cords when compared to reduced epithelium of the enamel organ. Odontogenic epithelial cells of some pericoronal follicles have proliferative potential, suggesting their association with the development of odontogenic lesions. The authors suggest that nonerupted, especially of the lower teeth, should be monitored and if necessary removed.

Molecular and classical chromosomal techniques reveal diversity in bushcricket genera of Barbitistini (Orthoptera)

The cytogenetic characteristics of 17 species of bushcricket belonging to eight genera of the tribe Barbitistini were examined by fluorescence in situ hybridization with 18S rDNA and (TTAGGn) telomeric as probes and by C-banding, silver, and fluorochrome staining. These markers were used to understand chromosomal organization and evolutionary relationships between genera or species within the same genus. The number of 18S rDNA clusters per haploid genome that co-localized with active nucleolus organizer regions (NORs) ranged from one to five, with the most common pattern being the presence of one NOR-bearing chromosome. This ribosomal cistron was preferentially located in the paracentromeric region of autosomes and very rarely in the sex chromosome. The results demonstrated coincidence between the localization of major ribosomal genes and active NORs and the position of C-band and GC-rich regions. The rDNA/NOR distribution and the composition of chromosome heterochromatin proved to be good cytogenetic markers for distinguishing species and phylogenetic lines and for understanding the genomic differentiation and evolution of Barbitistini. A comparison of cytogenetic and morphological or behavioral traits suggests that morphological and behavioral specialization in this group was not followed by major karyotype modification (except for Leptophyes). However, the occurrence and distribution of different repetitive DNA sites tends to vary among the taxa.

Ribosomal DNA is active in different B chromosome variants of the grasshopper Eyprepocnemis plorans

B chromosomes are considered to be genetically inert elements. However, some of them are able to show nucleolus organizer region (NOR) activity, as detected by both cytological and molecular means. The grasshopper Eyprepocnemis plorans shows a B chromosome polymorphism characterized by the existence of many B variants. One of them, B24, shows NOR activity in about half of B-carrying males in the Torrox population. Molecular data have suggested the recent origin for B chromosomes in this species, and on this basis it would be expected that NOR activity was widespread among the different B variants. Here we test this hypothesis in four different B chromosome variants (B1, B2, B5, and B24) from 11 natural populations of the grasshopper E. plorans covering the south and east of the Iberian Peninsula plus the Balearic Islands. We used two different approaches: (1) the cytological observation of nucleoli attached to the distal region of the B chromosome (where the rDNA is located), and (2) the molecular detection of the rDNA transcripts carrying an adenine insertion characteristic of B chromosome ITS2 sequences. The results showed NOR expression not only for B24 but also for the B1 and B2 variants. However, the level of B-NOR expression in these latter variants, measured by the proportion of cells showing nucleoli attached to the B chromosomes, was much lower than that previously reported for B24. This suggests the possibility that structural or genetic background conditions are enhancing the expressivity of the rDNA in the B24 variant.

Efficiency of cytogenetic methods in detecting a chromosome rearrangement induced by ionizing radiation in a cultivated chili pepper line (Capsicum baccatum var. pendulum – Solanaceae)

Purpose: To locate transient chromosome aberrations on a selected pepper cultivar and determine the tracing efficiency of different cytogenetic methods.Materials and methods: Seeds from Capsicum baccatum var. pendulum cultivar ‘Cayenne’ were treated with an acute dose of X-rays (300 Gy) and chromosome aberrations were analysed by different cytogenetic methods [Feulgen, silver staining for nucleolus organizer regions (silver positive nucleolus organizing regions or AgNOR), fluorescent banding, fluorescence in situ hybridization (FISH) and meiotic analysis].Results: A rearranged chromosome carrying two nucleolus organizing regions (NOR) induced by ionizing radiation was detected in the cultivar, with the occurrence of a small reciprocal exchange between a chromosome of pair no. 1 and another chromosome of pair no. 3, both carrying active NOR in short arms and associated chromomycin A positive/diamidino-phenylindole negative (CMA+/DAPI−) heterochromatin. Meiotic analysis showed a quadrivalent configuration, confirming a reciprocal translocation between two chromosomes.Conclusions: The use of X-rays in Capsicum allowed us to develop and identify a pepper line with structural rearrangements between two NOR-carrying chromosomes. We postulate that all the cytological techniques employed in this research were efficient in the search for chromosome aberrations. Particularly, Feulgen and AgNOR were the most suitable in those cases of transient rearrangements, whereas fluorescent banding and FISH were appropriate for intransitive ones.

Molecular cytogenetic map of the central bearded dragon, Pogona vitticeps (Squamata: Agamidae)

Reptiles, as the sister group to birds and mammals, are particularly valuable for comparative genomic studies among amniotes. The Australian central bearded dragon (Pogona vitticeps) is being developed as a reptilian model for such comparisons, with whole-genome sequencing near completion. The karyotype consists of 6 pairs of macrochromosomes and 10 pairs microchromosomes (2n = 32), including a female heterogametic ZW sex microchromosome pair. Here, we present a molecular cytogenetic map for P. vitticeps comprising 87 anchor bacterial artificial chromosome clones that together span each macro- and microchromosome. It is the first comprehensive cytogenetic map for any non-avian reptile. We identified an active nucleolus organizer region (NOR) on the sub-telomeric region of 2q by mapping 18S rDNA and Ag-NOR staining. We identified interstitial telomeric sequences in two microchromosome pairs and the W chromosome, indicating that microchromosome fusion has been a mechanism of karyotypic evolution in Australian agamids within the last 21 to 19million years. Orthology searches against the chicken genome revealed an intrachromosomal rearrangement of P. vitticeps 1q, identified regions orthologous to chicken Z on P. vitticeps 2q, snake Z on P. vitticeps 6q and the autosomal microchromosome pair in P. vitticeps orthologous to turtle Pelodiscus sinensis ZW and lizard Anolis carolinensis XY. This cytogenetic map will be a valuable reference tool for future gene mapping studies and will provide the framework for the work currently underway to physically anchor genome sequences to chromosomes for this model Australian squamate.

Comparative Cytogenetics among Populations ofHollandichthys multifasciatus(Teleostei: Characidae)

Two populations of Hollandichthys multifasciatus from headwaters of adjacent river coastal basins in Paraná state, southern Brazil, were cytogenetically studied in order to evaluate intraspecific divergences. Both populations presented 2n = 50 chromosomes, divided into 14m + 18sm + 18st in the sample from Antonina and 14m + 20sm + 16st for the population from Guaraqueçaba. Analyses of active nucleolar organizer regions (Ag-NORs) and fluorescent in situ hybridization (FISH) with 18S rDNA probes revealed a single metacentric pair bearing marks at proximal positions on the short arms. The location of the 5S rDNA and GC-rich sites showed chromosomal divergence between both populations. Therefore, several population markers were detected, reflecting a population differentiation, possibly driven by the formation of Paranaguá Bay and Serra do Mar mountain range. Since the existence of a species complex in H. multifasciatus has been previously characterized by morphological, molecular, and karyotype population differentiation, this species needs to be taxonomically studied in detail.

Identification of naturalized goat breeds under conservation from northeastern Brazil using chromosomal markers

In order to provide the first cytogenetic data of naturalized and threatened goat breeds from northeastern Brazil, cytogenetic analyses were carried out in individuals of Repartida and Moxotó breeds raised in Bahia and Ceará States. Males and females of both breeds had 2n = 60, with 29 autosomal acrocentric pairs plus the sex chromosome pair. The number of nucleolar organizer region (NOR)-bearing chromosomes ranged from 6 to 8 per metaphase in Moxotó and Repartida goats, respectively. The active NORs in Repartida individuals were located exclusively at the terminal regions of the long arms, as usually detected in Bovidae. Otherwise, Moxotó specimens presented a large autosomal pair with NORs on short arms. GC-rich heterochromatin was detected at the centromeres in both breeds, although polymorphic terminal C-bands were visualized on pair 25 in Moxotó. In addition, GC-rich regions were detected at the terminal regions of the long arms of a single pair in Repartida and of 20 chromosomes in Moxotó goats. The differences in both the number and/or position of Ag-NORs and GC-rich sites between Repartida and Moxotó breeds represent efficient cytogenetic markers that can be used in the identification and conservation of the genetic integrity of each lineage. In spite of the small effective population size of these breeds, chromosomal abnormalities related to drift or inbreeding effects were absent in the samples analyzed.

Exploring the mechanisms of alcohol-related damage in oral mucosa – is oxidative stress associated with the increase in cell proliferation in rat tongue epithelium?

Context: Alcohol consumption has been related to a cell proliferation increase in oral epithelium but its mechanism remains unclear.Objective: The aim of this study was to investigate whether oxidative stress parameters are implicated in the induction of cell proliferation in rat tongue epithelium after different times of chronic alcohol consumption.Materials and methods: Cell proliferation was assessed in tongue epithelium using AgNOR (argyrophilic proteins related to active nucleolar organizer regions) quantification. Oxidative stress parameters [lipid peroxidation, protein carbonyls, superoxide dismutase activity and catalase (CAT) activity and immunocontent] and Nrf2 immunocontent were quantified in tongue homogenates.Results and discussion: Mean AgNOR numbers (mAgNOR) per nucleus was 2.22 ± 0.30 in ventral tongue epithelium after 120 days of alcohol consumption (vs. 1.87 ± 0.18 for control animals and 1.91 ± 0.23 for animals treated with alcohol for 60 days) indicating cell proliferation increase (p < 0.05, ANOVA followed by Tukey post hoc). Interestingly, 60 days of alcohol consumption induced changes in oxidative stress parameters, but no alteration in cell proliferation. Vitamin E co-treatment was conduced in order to evaluate its possible protective effects. The 120 day Tween + vitamin E + alcohol treatment induced an increase in mAgNORs when compared to the Tween + vitamin E treated group (respectively 2.10 ± 0.30 vs. 1.77 ± 0.11, p < 0.05, ANOVA followed by Tukey post hoc), showing that vitamin E co-treatment had no protective effects. In addition, an inverse association was observed between CAT activity and AgNORs quantity (R = −0.32; p < 0.05, Person’s correlation) as well as the possible involvement of Nrf2 in alcohol-related damage.Conclusions: Our findings suggest that the increase in cell proliferation associated with alcohol-related damage has no direct relation with an imbalance in oxidative parameters. In contrast, our results indicate that hydrogen peroxide may be implicated in cellular signaling during proliferation in the oral mucosa.