Bleomycin (BLM, CAS No. 9041-93-4) is a well-established antitumor and antibiotic agent commonly used as a positive control in genotoxicity assays due to its ability to induce mutagenic effects across multiple organisms. In mammalian cells, BLM induces chromosomal damage, in both chromatids and chromosomes [1,2]. Cytogenetic studies have demonstrated that BLM induces chromosome-type aberrations, such as dicentric and multicentric chromosomes, when acting on cells in the G0 phase. Conversely, in the G2 phase, BLM predominantly causes chromatid-type aberrations, with gaps and breaks being the most frequently observed abnormalities. Beyond these primary effects, the BLM can induce complex chromosomal exchanges involving multiple breaks across two or more chromosomes. The genotoxic mechanism of BLM is mediated by free radicals, classifying it as a radiomimetic agent. Notably, BLM-induced dicentric chromosomes in human lymphocytes exhibit a linear dose-response relationship, akin to the effects observed with ionizing radiation [[1], [2], [3], [4]]. Furthermore, BLM is well-characterized for its ability to bind and damage DNA through mechanisms such as base oxidation, alkali-labile lesions, and DNA cleavage. Upon cellular entry, BLM is activated by reduced transition metals (e.g., Fe2+ or Cu+) and oxygen, facilitating the removal of the 4′-hydrogen atom from the deoxyribose moiety of pyrimidines. This reaction generates 4′-radical intermediates, leading to apurinic/apyrimidinic (AP) sites or gapped DNA with 3′-phosphoglycolate/5′-phosphate termini. Subsequent incision of the complementary strand results in double-strand breaks (DSBs), which are lethal if not properly repaired. BLM-induced DNA damage exhibits sequence specificity, preferentially targeting 5′-GT∗ and 5′-GC∗ dinucleotides (∗indicating the cleaved nucleotide). Consequently, chromatin structure significantly influences the extent of DNA damage caused by BLM. Transcriptional activity has also been identified as a factor that enhances susceptibility to BLM-induced DNA damage [5,6].

In this context, an intriguing question arises regarding the response to BLM-induced genotoxic insult in genomes that exhibit pronounced structural differences compared to the human genome, which may account for differential genomic stability. To address this question, species of non-human primates represent suitable models, as their genomes share substantial sequence homology with humans while retaining distinctive structural differences.

The genome is constantly subjected to damage from exogenous factors (e.g., radiation, chemicals) and endogenous processes (e.g., reactive oxygen species). Fragile sites, specific chromosomal loci prone to breaks and rearrangements under certain conditions, are a cytogenetic manifestation of DNA damage. These sites are linked to genetic diseases and recurrent rearrangements, highlighting the relationship between genome instability and genome structure. Ruiz-Herrera et al. [7] characterized chromosomal segments involved in primate evolution based on conserved chromosomal syntenies relative to the human karyotype and an inferred ancestral primate karyotype. This work identified unstable chromosomal bands involved in evolutionary reorganizations.

The genomes of primates exhibit a striking characteristic: a high abundance of repetitive sequences, predominantly satellite DNA (satDNA) [8]. These sequences constitute a significant component of heterochromatin blocks, which serve as structural elements defining chromosomes. Heterochromatin plays an essential role in maintaining genome integrity by contributing to processes such as chromosome segregation, telomere protection, transposon activity suppression, and DNA repair mechanisms [9]. Genomic integrity is crucial for both clinical outcomes and evolutionary chromosomal rearrangements.

Within Platyrrhini, the Sapajus and Cebus genera (capuchin monkeys, Cebidae) stand out due to their genomes containing approximately 8–15 % heterochromatin—an unusually high proportion compared to other platyrrhines—and its remarkable conservation of euchromatic regions relative to the human karyotype [[10], [11], [12]]. These distinctive genomic traits underscore the experimental relevance of Sapajus in studies of chromosomal evolution and genome stability. Notably, heterochromatin plays a significant role in the speciation process within platyrrhines, particularly in capuchin monkeys. Classical cytogenetic techniques, including C-banding, DAPI-CMA3 staining, and molecular cytogenetics (e.g., FISH), have revealed extensive karyotypic diversity both within and among species. In these monkeys, the chromosome number (2n) does not vary greatly (2n = 52–54); however, this diversity is characterized by structural chromosomal rearrangements, such as fusions, fissions, and inversions, which are frequently associated with heterochromatin regions [12]. Furthermore, these studies have documented significant polymorphisms in band quantity, size, distribution, and karyotypic localization [13] (Fig. 1A and B). In addition, heterochromatin composition was investigated using the DAPI-CMA3 fluorescent banding technique allowing for the determination of the proportion of adenine-thymine (AT) or guanine-cytosine (GC) base pairs within heterochromatic regions. Specifically, AT-rich genomic regions exhibit strong DAPI signals, while GC-rich regions show positive CMA3 signals [14]. DAPI-CMA3 characterization of Sapajus species revealed that DAPI-positive signals did not exhibit a consistent pattern with other banding techniques. In contrast, CMA3-positive bands consistently co-localized with heterochromatin blocks, indicating that heterochromatin in this genus is predominantly GC-rich [12] (Fig. 1C).

Recent investigations have highlighted the relationship between genomic stability, rearrangement hotspots, and their evolutionary significance. For instance, in Sapajus cay (2n = 54, XX/XY), a low frequency of sister chromatid exchanges (SCEs) was observed predominantly near heterochromatin blocks (e.g., on chromosome pairs 4, 6, 11, 12, 13, 17, and 19) or in conserved karyotypic regions (Fig. 1A). The substantial proportion of heterochromatin in the Sapajus genome may act as a stabilizing and protective factor against genotoxic damage [16,15]. This genomic composition suggests the presence of regions with differential fragility, prone to reorganization, alongside conserved tracts with varying levels of evolutionary plasticity. Given the critical role of heterochromatin in genome stability, it becomes essential to explore the specific DNA damage response mechanisms in Sapajus, analyzing them across various analytical levels, including cytological and cytomolecular perspectives. To achieve this goal, in this study, peripheral blood lymphocytes from Sapajus cay were exposed to BLM. In vitro Chromosomal aberration (CA) assay and FISH with centromere and telomere specific probes were applied to analyze the genomic stability of Sapajus cay in response to genotoxic stress.