Shambhu K Roy

Spontaneous Chromosomal Instability in Breast Cancer Families

Spontaneous chromosomal instability has been correlated with cancer predisposition. In the present study, the phenomenon has been evaluated using two cytogenetic markers, namely, frequency of spontaneous sister chromatid exchanges (SCE) and spontaneous chromosomal aberrations (CA) in peripheral blood lymphocytes of hereditary breast cancer (HBC) patients (n = 11) and healthy blood relatives (HBR, n = 36). A statistically significant difference was observed for both the endpoints between HBC patients and controls (P < 0.001), HBC patients and HBR (P < 0.001), as well as HBR and controls (P < 0.001). Thus, 63.64% of the HBC patients and 25% of HBR showed a mean CA/cell value higher than the highest mean CA/cell value of the controls (0.11 CA/cell). Similarly, 81.81% of the HBC patients and 61.11% of HBR showed a mean SCE/cell value higher than the highest mean SCE/cell value of the controls (9.60 SCE/cell). Chromosomal aberrations were more frequently observed in the B and E group of chromosomes in HBC patients and HBR. These findings primarily indicate the high level of chromosomal instability in breast cancer families, and might be one of the predisposing factors for high risk of cancer in HBR.

Integration of Pig-a, micronucleus, chromosome aberration and comet assay endpoints in a 28-day rodent toxicity study with urethane

As part of the international Pig-a validation trials, we examined the induction of Pig-a mutant reticulocytes and red blood cells (RET(CD59-) and RBC(CD59-), respectively) in peripheral blood of male Sprague Dawley(®) rats treated with urethane (25, 100 and 250mg/kg/day) or saline by oral gavage for 29 days. Additional endpoints integrated into this study were: micronucleated reticulocytes (MN-RET) in peripheral blood; chromosome aberrations (CAb) and DNA damage (%tail intensity via the comet assay) in peripheral blood lymphocytes (PBL); micronucleated polychromatic erythrocytes (MN-PCE) in bone marrow; and DNA damage (comet) in various organs at termination (the 29th dose was added for the comet endpoint at sacrifice). Ethyl methanesulfonate (EMS; 200mg/kg/day on Days 3, 4, 13, 14, 15, 27, 28 and 29) was evaluated as the concurrent positive control (PC). All animals survived to termination and none exhibited overt toxicity, but there were significant differences in body weight and body weight gain in the 250-mg/kg/day urethane group, as compared with the saline control animals. Statistically significant, dose-dependent increases were observed for urethane for: RET(CD59-) and RBC(CD59-) (on Days 15 and 29); MN-RET (on Days 4, 15 and 29); and MN-PCE (on Day 29). The comet assay yielded positive results in PBL (Day 15) and liver (Day 29), but negative results for PBL (Days 4 and 29) and brain, kidney and lung (Day 29). No significant increases in PBL CAb were observed at any sample time. Except for PBL CAb (likely due to excessive cytotoxicity), EMS-induced significant increases in all endpoints/tissues. These results compare favorably with earlier in vivo observations and demonstrate the utility and sensitivity of the Pig-a in vivo gene mutation assay, and its ability to be easily integrated, along with other standard genotoxicity endpoints, into 28-day rodent toxicity studies.

Mitomycin C induced chromosomal aberrations in young cancer patients

Mitomycin-C (MMC) induced Chromosomal aberration (CA) frequencies were studied in 48 h peripheral blood lymphocyte (PBL) cultures of untreated cancer patients of young age (maximum age 12 years, n=77). Control population (n=71) consisted of age-matched group (maximum age 12 years, n=21); elder controls (minimum age 60 years, n=19) and healthy first degree relatives, i.e., parents or siblings of the pediatric cancer patients (mean age 24.3 years, n=31) as they share their genome and environment. Induced CA levels were found to be significantly higher among pediatric cancer patients as compared to control groups. The age-matched and elder control groups showed comparable CA levels. The first degree relatives controls showed higher induced CA levels as compared to pediatric and elder control groups. The present results indicate that there are different degrees of mutagen sensitivity prevailing in normal population. This may be responsible for differential cancer proneness. High degree of mutagen sensitivity in cancer patients may also be playing a major role in cancer onset at an early age.

Cytogenetic evaluation of 20 sporadic breast cancer patients and their first degree relatives

Many genetic abnormalities disclosed even in somatic cells like peripheral blood lymphocytes may mark footprint(s) of malignancy(ies). The present cytogenetic study on peripheral blood lymphocytes of sporadic breast cancer patients (n = 20) and their first degree relatives (n = 39) reports abnormalities of chromosomes 16, 5, 12, and 17 respectively in 17.59%, 8.33%, 6.48%, and 5.57% cells of patients and 15.83%, 8.33%, 7.5%, and 5% cells of their first degree relatives. These common chromosomal abnormalities pave the way to assume why first degree relatives of sporadic breast cancer patients are at increased risk of developing the same or other malignancies.

Emerging Approaches in Predictive Toxicology

Predictive toxicology plays an important role in the assessment of toxicity of chemicals and the drug development process. While there are several well‐established in vitro and in vivo assays that are suitable for predictive toxicology, recent advances in high‐throughput analytical technologies and model systems are expected to have a major impact on the field of predictive toxicology. This commentary provides an overview of the state of the current science and a brief discussion on future perspectives for the field of predictive toxicology for human toxicity. Computational models for predictive toxicology, needs for further refinement and obstacles to expand computational models to include additional classes of chemical compounds are highlighted. Functional and comparative genomics approaches in predictive toxicology are discussed with an emphasis on successful utilization of recently developed model systems for high‐throughput analysis. The advantages of three‐dimensional model systems and stem cells and their use in predictive toxicology testing are also described. Environ. Mol. Mutagen. 55:679–688, 2014.

Bimolane induces multiple types of chromosomal aberrations in human lymphocytes in vitro.

Bimolane has been commonly used in China for the treatment of psoriasis and various types of cancer. Patients treated with bimolane have been reported to have an increased risk of developing therapy-related leukemias. Although bimolane has been identified as a human leukemia-inducing agent, little is known about its genotoxic effects, and a systematic study of the types of chromosomal alterations induced by this compound has not been performed. In this study, a combination of immunochemical, molecular and conventional cytogenetic techniques has been used to study the chromosomal alterations induced by bimolane in cultured human lymphocytes. Immunochemical staining with the CREST antibody indicated that bimolane induces micronuclei (MN) originating primarily from chromosome breakage. Interestingly fluorescence in situ hybridization (FISH) with differentially labeled chromosomes 1 and 9 centromeric probes indicated that bimolane also caused non-disjunction and polyploidy. Consistent with this, an expedited analysis of Giemsa-stained metaphase chromosomes in bimolane-treated lymphocytes revealed a high frequency of polyploidy/hyperdiploidy as well as dicentric chromosomes, and premature centromeric division (PCD). In addition, bimolane was also found to produce binucleated cells, possibly through an interference with normal functioning of intermediate filaments. As a follow-up to these studies, three different types of commercially available bimolane formulations obtained from different Chinese manufacturers were also evaluated. The effects seen with the formulated bimolane were similar to those seen with the synthesized compound. Our studies indicate that bimolane effectively induces a variety of cellular and chromosomal changes in cultured lymphocytes and that similar alterations occurring in bone marrow stem cells could contribute to the development of the secondary cancers seen in bimolane-treated patients.

The EpiDerm™ 3D human reconstructed skin micronucleus (RSMN) assay: Historical control data and proof of principle studies for mechanistic assay adaptations

The in vitro human reconstructed skin micronucleus (RSMN) assay in EpiDerm™ is a promising novel animal alternative for evaluating genotoxicity of topically applied chemicals. It is particularly useful for assessing cosmetic ingredients that can no longer be tested using in vivo assays. To advance the use of this test especially for regulatory decision-making, we have established the RSMN assay in our laboratory according to Good Laboratory Practice and following the principles of the OECD test guideline 487 in vitro mammalian cell micronucleus test. Proficiency with the assay was established by correctly identifying direct-acting genotoxins and genotoxins requiring metabolism, as well as non-genotoxic/non-carcinogenic chemicals. We also report the analysis of our historical control data that demonstrate vehicle control and positive control values for %micronuclei in binucleated cells are in the ranges reported previously. Technical issues including evaluating various solvents with both 48h and 72h treatment regimens were investigated. For the first time, mechanistic studies using CREST analysis revealed that the RSMN assay is suitable for distinguishing aneugens and clastogens. Moreover, the assay is also suitable for measuring cytokines as markers for proliferative and toxic effects of chemicals.

Chromosomal Aberrations in Young Cancer Patients

Spontaneous level of chromosomal aberrations (CA) is considered to be indicative of inherent cancer predisposition, which plays a major role in total cancer incidence. We have studied spontaneous CA levels in in vitro cultured peripheral blood lymphocytes of pediatric cancer patients (n = 77). Results were compared with those of control subjects (n = 72), including: age-matched controls; elder controls (minimum age 60 years); and healthy first-degree relatives (FDR) of pediatric cancer patients. Pediatric cancer patients showed the highest mean CA/cell value, which was statistically significant as compared to their age-matched counterparts, elder controls, and the FDRs. As compared to 7% of all the three control groups collectively, 32.4% of pediatric cancer patients showed > 0.1 mean CA/cell value. One of the FDRs with a very high frequency of CA developed cancer within three years. The results suggest that spontaneous levels of chromosomal aberrations may be used as one of the biomarkers for cancer predisposition study.

Analysis of historical negative control group data from the in vitro micronucleus assay using human lymphocytes

A database of the micronuclei counts was built up for historical negative control data from human lymphocyte in vitro micronuclei tests (MnVit) carried out in 8 laboratories with experience of the method. The mean incidence of micronucleated cells (mnt)/1000 cells ranged from 2.2/1000 to 15.9/1000. There were no large differences in incidence between the presence or absence of S9 mix or between different treatment lengths. There was also little evidence that different solvents affected the numbers of micronuclei appreciably. A number of laboratories did show significant inter-experiment variability, indicating that there remained unidentified factors affecting frequencies. Donor variance may be one such factor. Inter-individual variability may explain some of these differences. The approximate 7.5-fold difference in mnt/1000 scores in a relatively small group of experienced laboratories illustrates the potential complications that can arise if a metric like a fold increase was considered the only biologically important finding. Although there is inherent variability between experiments, it was evident that within a laboratory the overall laboratory mean remains constant over time. It is believed that these findings will provide help to laboratories conducting studies using human lymphocytes in the MnVit and to those involved in the assessment of MnVit results.

Pentasomy 21 as a sole abnormality in an atypical CML patient in chronic phase

Case report of a translocation : Pentasomy 21 as a sole abnormality in an atypical CML patient in chronic phase.