An alternative approach of TUNEL assay to specifically characterize DNA fragmentation in cell model systems.

DNA damage is one of the most important effects induced by chemical agents. We report a comparative analysis of DNA fragmentation on three different cell lines using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, generally applied to detect apoptosis. Our approach combines cytogenetic techniques and investigation in detached cellular structures, recovered from the culture medium with the aim to compare the DNA fragmentation of three different cell line even beyond the cells adherent to substrate. Consequently, we detect any fragmentation points on single chromosomes, whole nuclei and other cellular structures. Cells were exposed to resveratrol (RSV) and doxorubicin (Doxo), in single and combined treatments. Control and treated astrocytes showed DNA damage in condensed nuclei and detached structures. Caco-2 cells showed fragmented DNA only after Doxo-treatment, while controls showed fragmented chromosomes, indicating DNA damage in replicating cells. MDA-MB-231 cells showed nuclear condensation and DNA fragmentation above all after RSV-treatment and related to detached structures. This model proved to perform a grading of genomic instability (GI). Astrocytes show a hybrid level of GI. Caco-2 cells showed fragmented metaphase chromosomes, proving that the DNA damage was transmitted to the daughter cells probably due to an absence of DNA repair mechanisms. Instead, MDA-MB-231 cells showed few or no fragmented metaphase, suggesting a probable activation of DNA repair mechanisms. By applying this alternative approach of TUNEL test, we obtained data that can more specifically characterize DNA fragmentation for a suitable application in various fields.

Investigation of the protective effects of piceatannol on experimental subarachnoid hemorrhage in rats.

BACKGROUND: Subarachnoid hemorrhage (SAH) is one of the most prevalent brain injuries in humans which has poor prognosis and high mortality rates. Due to several medical or surgical treatment methods, a gold standard method doesn't exist for SAH treatment. Piceatannol (PCN), a natural analog of resveratrol, was reported to reduce inflammation and apoptosis promising a wide range of therapeutic alternatives. In this study, we aimed to investigate the effects of PCN in an experimental SAH model. The alleviating effects of PCN in the hippocampus in an experimental SAH model were investigated for the first time. METHODS AND RESULTS: In this study, 27 Wistar Albino male rats (200-300 g; 7-8 week) were used. Animals were divided into three groups; SHAM, SAH, and SAH + PCN. SAH model was created with 120 µl of autologous arterial tail blood to prechiasmatic cisterna. 30 mg/kg PCN was administered intraperitoneally at 1st h after SAH. Neurological evaluation was performed with Garcia's score. RT-PCR was performed for gene expression levels in the hippocampus. Pyknosis, edema, and apoptosis were evaluated by H&E and TUNEL staining. Our results indicated that PCN administration reduced apoptosis (P < 0.01), cellular edema, and pyknosis (P < 0.05) in the hippocampus after SAH. Moreover, PCN treatment significantly decreased the expression levels of TNF-α (P < 0.01), IL-6 (P < 0.05), NF-κB (P < 0.05), and Bax (P < 0.05) in the hippocampus. CONCLUSIONS: Our results demonstrated that PCN might be a potential therapeutic adjuvant agent for the treatment of early brain injury (EBI) following SAH. Further studies are required to clarify the underlying mechanisms and treatment options of SAH.

Hepatic inflammation, ballooning, and pyknosis caused by LED light exposure in a mouse model, with differential effects by age and gender.

Light-emitting diode (LED) is commonly used in lighting and digital devices in modern life, which delivers higher levels of blue light than other light sources. Previous work indicated that exposure to blue lights increases serum oxidative stress and affects hepatic functions in animals. However, the detailed hepatic pathogenesis caused by blue lights remains largely elusive. This study investigated the characteristics of hepatic injuries caused by LED light exposure in a mouse model. C57BL/6 mice were exposed the LED lights at 1000 lux, 12 h per day for 45 days or at 4500 lux, 1 h per day for 7 days. The mice were aged 8 weeks or 36 weeks in both genders and maintained under a 12 h light/dark cycle without alteration of diet pattern. Liver tissue sections were obtained for hematoxylin and eosin (H&E) and immunohistochemical staining. The mice with 1000 lux exposure displayed severe liver injuries, including inflammation, ballooning, and pyknosis, which were found to a lesser extent in the 4500 lux mice, and aging aggravated the hepatic injuries. The hepatocellular ballooning was found more severe in the males than the females. In contrast, the females expressed the F4/80 and TNF-α inflammatory markers more evidently. Taken together, LED light exposure may have detrimental effects on liver health, particularly in vulnerable groups such as the elderly and the females with excessive exposure to LED lights, even if they maintain a normal diet and regular light/dark cycles. The potential risk should be considered by both the clinicians and the public.

Saffron stigma extract and crocin play an important neuroprotective role in therapeutic measures against benzo[a]pyrene-induced behavioral alterations in zebrafish.

Saffron is a well-known expensive spice, which has many pharmacological properties against a variety of ailments. Saffron stigma and leaf contain apocarotenoids and bioactive phytochemicals having therapeutic potential against human disorders. Polycyclic aromatic hydrocarbons (PAHs) are one of the most common toxins in today's aquatic environment. Benzo[a]pyrene (B[a]P), a high molecular weight PAHs prototype, and reported as a potent neurotoxicant, which is profoundly contaminating the environment. The present study investigated the therapeutic efficacy of Saffron stigma extracts and crocin, on B[a]P-induced behavioral changes, altered antioxidant activities, and neurodegeneration in zebrafish. The behavioral responses monitored through the light-dark preference test and novel tank diving test suggested that B[a]P treated zebrafish group showed alteration in anxiolytic-like behavior. Animals exhibited their native behavior when treated alone with Saffron Stigma Extract (SSE) and crocin, an apocarotenoid which also reduced the altered behavior induced by B[a]P. The SSE and crocin stimulated the antioxidant activities with an accumulation of reduced glutathione and catalase enzymes, indicating a protective role against B[a]P-induced oxidative stress and behavioral deficits. The histopathological studies showed the percentage change of pyknotic cell counts in the Periventricular Gray Zone region of the Optic Tectum was 1.74 folds high in B[a]P treated animals as compared to control. Furthermore, the treatment of SSE and crocin reduced the pyknosis process induced by B[a]P-mediated neurodegeneration, possibly due to a better protective mechanism. Future studies may reveal the detailed mechanisms of action of potent SSE and crocin like bioactive compounds having neuroprotective potentials against neurodegenerative diseases.

TcdB of Clostridioides difficile Mediates RAS-Dependent Necrosis in Epithelial Cells.

A Clostridioides difficile infection (CDI) is the most common nosocomial infection worldwide. The main virulence factors of pathogenic C. difficile are TcdA and TcdB, which inhibit small Rho-GTPases. The inhibition of small Rho-GTPases leads to the so-called cytopathic effect, a reorganization of the actin cytoskeleton, an impairment of the colon epithelium barrier function and inflammation. Additionally, TcdB induces a necrotic cell death termed pyknosis in vitro independently from its glucosyltransferases, which are characterized by chromatin condensation and ROS production. To understand the underlying mechanism of this pyknotic effect, we conducted a large-scale phosphoproteomic study. We included the analysis of alterations in the phosphoproteome after treatment with TcdA, which was investigated for the first time. TcdA exhibited no glucosyltransferase-independent necrotic effect and was, thus, a good control to elucidate the underlying mechanism of the glucosyltransferase-independent effect of TcdB. We found RAS to be a central upstream regulator of the glucosyltransferase-independent effect of TcdB. The inhibition of RAS led to a 68% reduction in necrosis. Further analysis revealed apolipoprotein C-III (APOC3) as a possible crucial factor of CDI-induced inflammation in vivo.

Thermal stress induces pyknosis in pigeon erythrocytes: digital image analysis.

Pyknosis or hypercondensation of chromatin is informative in the understanding of nucleosomal packing in translationally inactive chromatin and in the compression of cell death. However, mechanisms that result in the formation of avian erythrocytes with variant nuclear morphology are poorly understood.Purpose: In this work, we evaluated pyknosis in pigeon erythrocytes treated with thermal stress using Digital Image Analysis (DIA).Materials and methods: Pigeon erythrocytes were treated at thermal stress (33 °C, 43 °C, and 53 °C), and nuclear modifications were analyzed by DIA.Results: Our results showed that thermal stress induced DNA condensation. Based on DNA fluorescent staining and compaction, four subclasses with progressively more pyknotic nuclei each could be distinguished. Alkaline comet assay showed that the presence of pyknotic nuclei was associated with the DNA fragmentation typical of apoptosis. DIA analysis showed a decrease of nuclear area and a significant increase of fluorescence intensity with respect to non-pyknotic nucleus. Additionally we observed nuclear dissolution events associated with swell and loose membrane integrity.Conclusion: These findings can contribute to the evaluation of health and metabolic status in diagnostic cytology, especially in neoplastic conditions and infection by microorganisms.

Necrotic pyknosis is a morphologically and biochemically distinct event from apoptotic pyknosis.

Classification of apoptosis and necrosis by morphological differences has been widely used for decades. However, this usefulness of this method has been seriously questioned in recent years, mainly due to a lack of functional and biochemical evidence to interpret the morphology changes. To address this matter, we devised genetic manipulations in Drosophila to study pyknosis, a process of nuclear shrinkage and chromatin condensation that occurs in apoptosis and necrosis. By following the progression of necrotic pyknosis, we surprisingly observed a transient state of chromatin detachment from the nuclear envelope, followed by the nuclear envelope completely collapsing onto chromatin. This phenomenon led us to discover that phosphorylation of barrier-to-autointegration factor (BAF) mediates this initial separation of nuclear envelope from chromatin. Functionally, inhibition of BAF phosphorylation suppressed necrosis in both Drosophila and human cells, suggesting that necrotic pyknosis is conserved in the propagation of necrosis. In contrast, during apoptotic pyknosis the chromatin did not detach from the nuclear envelope and inhibition of BAF phosphorylation had no effect on apoptotic pyknosis and apoptosis. Our research provides the first genetic evidence supporting a morphological classification of apoptosis and necrosis through different forms of pyknosis.

Expression of the human TRIM14 and its mutant form (P207L) promotes apoptosis in transgenic loaches.

The tripartite-motif (TRIM)14 protein, one of the TRIM family members, was shown to participate in the antiviral and antibacterial defence. Besides, it appears to play an essential role in the processes of oncogenesis. In some types of human tumour cells, TRIM14 has been shown to inhibit apoptosis, while in others-the overexpression of TRIM14 promotes apoptosis. However, whether TRIM14 mediates apoptosis in the normal cells remains unknown. In the present study, we investigated the possible participation of the human TRIM14 gene and its mutant form (620C > T) in the induction of apoptosis in the transgenic larvae loach Misgurnus fossilis L. We observed that the expression of both forms of TRIM14 gene was accompanied by the increase of the frequency of pyknotic nuclei in fish embryos compared to control groups. Accordingly, using the TUNEL assay, the enhanced apoptosis was revealed upon expression of both forms of TRIM14 gene. The transcription of proapoptotic genes (bax, tp53, and casp9) was significantly increased in transgenic loaches expressing human wild-type TRIM14, but remained unchanged upon expression of its mutant form. In addition, the transcription of c-myc was upregulated in transgenic loaches expressing both forms. Thus, it can be assumed that during embryonic development TRIM14 has a proapoptotic effect on the cells via the activation of c-myc, tp53, and bax genes. Apparently, the mutant TRIM14 directs apoptosis via c-myc by p53-independent mechanism.

Glucosyltransferase-dependent and -independent effects of TcdB on the proteome of HEp-2 cells.

Toxin B (TcdB) of the nosocomial pathogen C. difficile has been reported to exhibit a glucosyltransferase-dependent and -independent effect on treated HEp-2 cells at toxin concentration above 0.3 nM. In order to investigate and further characterize both effects epithelial cells were treated with wild type TcdB and glucosyltransferase-deficient TcdBNXN and their proteomes were analyzed by LC-MS. Triplex SILAC labeling was used for quantification. Identification of 5212 and quantification of 4712 protein groups was achieved. Out of these 257 were affected by TcdB treatment, 92 by TcdBNXN treatment and 49 by both. TcdB mainly led to changes in proteins that are related to "GTPase mediated signaling" and the "cytoskeleton" while "chromatin" and "cell cycle" related proteins were altered by both, TcdB and TcdBNXN . The obtained dataset of HEp-2 cell proteome helps us to better understand glucosyltransferase-dependent and -independent mechanisms of TcdB and TcdBNXN , particularly those involved in pyknotic cell death. All proteomics data have been deposited in the ProteomeXchange with the dataset identifier PXD006658 (https://proteomecentral.proteomexchange.org/dataset/PXD006658).

Transmission electron microscopic analysis of malathion-induced cytotoxicity in granulosa cells of caprine antral follicles.

Malathion, one of the most abundantly used organophosphate pesticides, has immoderate potency as a cytotoxic and genotoxic compound that induces toxicity in granulosa cells, resulting in its apoptosis. Thus, the present study aims to employ ultrastructural analysis for assessing the cytotoxicity of malathion at nanomolar concentrations (1 nM and 10 nM) in granulosa cells of caprine antral follicles at different exposure durations. Transmission electron microscopy revealed diminished cell-cell contact and cellular integrity, presence of crescent-shaped nucleus, chromatin condensation, and pyknosis with nuclear membrane folding, accumulation of lipid droplets with occurrence of cytoplasmic protrusions in granulosa cells treated with 1 nM malathion, whereas at 10 nM concentration, along with apoptotic attributes, prominent association of nucleus, endoplasmic reticulum, mitochondria and lipid droplets, nucleus invagination into lipid droplets, apical localization of lipid bodies, and occurrence of autophagic body were observed as compared to healthy granulosa cells in control with normal intact cellular integrity, well-developed cellular association, and doubled membrane nuclear lamina with homogenously dispersed chromatin surrounded by intact mitochondria with well-developed cristae. Thus, the results of ultrastructural analysis clearly suggest that nanomolar concentration of malathion induces apoptotic hallmarks within the granulosa cells of antral follicles that play a consequential role in increasing the incidence of follicular atresia, thereby affecting the overall fertility.

Granulosa cell apoptosis by impairing antioxidant defense system and cellular integrity in caprine antral follicles post malathion exposure.

Toxicological studies have demonstrated the exposure-risk relationship of several pesticides on reproduction of living organisms. To evaluate the role of malathion as a reproductive toxicant, this study aims at assessing the cytological and biochemical changes in the granulosa cells after malathion exposure in dose (1 nM, 10 nM, 100 nM) and time (4 h, 6 h, 8 h) dependent manner. Histomorphological analysis, fluorescence assay, apoptosis quantification, and terminal deoxynucleotidyl transferase d-UTP mediated nick end labeling (TUNEL) assay were done to determine cytological changes, whereas antioxidant enzyme assays were done to measure the oxidative stress in malathion treated ovarian antral follicles. Histological studies exhibited the occurrence of highly condensed or marginated chromatin with fragmented nucleus, pyknosis, loss of membrane integrity, increased empty spaces, and vacuolization in malathion treated granulosa cells. Ethidium bromide/acridine orange (EB/AO) fluorescence staining demonstrated a significant increase in incidence and percentage of apoptosis after malathion exposure (p < 0.001), both between and within the groups. Malathion exposure also resulted in increased DNA fragmentation and decline in both antioxidant enzymes activity namely catalase (CAT) and superoxide dismutase (SOD) in granulosa cells of antral follicles. Moreover, there was found a significant negative correlation between the apoptosis incidence and the level of antioxidant enzymes activity, SOD (r = -0.73 p < 0.01) and CAT (r = -0.80 p < 0.01), in malathion treated ovarian antral follicles. Thus, highlighting the role of DNA fragmentation and declining antioxidant level as a possible mechanism underlying malathion induced reproductive toxicity. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1944-1954, 2016.

Atomic force microscopic investigation of respiratory syncytial virus infection in HEp-2 cells.

Respiratory syncytial virus (RSV) primarily causes bronchiolitis and pneumonia in infants. In spite of intense research, no safe and effective vaccine has been developed yet. For understanding its pathogenesis and development of anti-RSV drugs/therapeutics, it is indispensable to study the RSV-host interaction. Although, there are limited studies using electron microscopy to elucidate the infection process of RSV, to our knowledge, no study has reported the morphological impact of RSV infection using atomic force microscopy. We report the cytoplasmic and nuclear changes in human epidermoid cell line type 2 using atomic force microscopy. Human epidermoid cell line type 2 cells, grown on cover slips, were infected with RSV and fixed after various time periods, processed and observed for morphological changes using atomic force microscopy. RSV infected cells showed loss of membrane integrity, with degeneration in the cellular content and cytoskeleton. Nuclear membrane was disintegrated and nuclear volume was decreased. The chromatin of the RSV infected cells was condensed, progressing towards degeneration via pyknosis and apoptosis. Membrane protrusions of ~150-200 nm diameter were observed on RSV infected cells after 6 h, suggestive of prospective RSV budding sites. To our knowledge, this is the first study of RSV infection process using atomic force microscopy. Such morphological studies could help explore viral infection process aiding the development of anti-RSV therapies.

Microplastics induced ileum damage: Morphological and immunohistochemical study.

Microplastics (MPs) are small pieces of plastic that are widely distributed in the environment and accumulate within living organisms, so they are the most common types of pollutants at the present time. One of the most widespread types of MP in the environment is polyethylene (PE) MPs. There have been many published studies on the effect of PE MPs combined with other pollutants or chemicals such as benzoanthracene, emamectin benzoate, heavy metals and 4-nonylphenol, on some marine, amphibian, and mouse models. However, research has rarely been conducted on how single-use PE MPs affect the ileum of mammals. The current study is focused on the impact of PE MP exposure with different concentration (6, 60, 600 µg/mL PE/MPs) for 15 days, followed by 15 days of recovery on small intestine(ileum) of C57BL/6 murine model with precision and detail at the cell level by using different technique (histology, histochemistry, immunohistochemistry, and transmission electron microscope). Results demonstrated that the intestinal tissue exhibited nuclear pyknosis, villus deformation, shortness of villi, degeneration of lamina propria, hyperplasia of goblet cells, increase of goblet cells secretion, Alcian blue and Periodic acid-Schiff stain positivity of intact goblet cells, highly significance of P53 immunoreaction expression specially in high concentrations (600 µg/day of PE/MPs) and Ki-67 immunoreaction expression. RESEARCH HIGHLIGHTS: Different doses of microplastics (MPs) induced sever morphological alternations and clinical observations. MPs were deposits in cells and were observed in ultrastructure study. Recovery period able to ameliorate to the most extent the alternations caused by MPs administration.

Evaluation of Histomorphological Changes in Breast Cancer Post-Neoadjuvant Chemotherapy.

Breast cancer, a leading cause of global female mortality, demands comprehensive diagnostic and therapeutic strategies. This study delves into the nuanced realm of post-neoadjuvant chemotherapy breast cancer specimens, emphasizing the imperative need for pathologists to discern stromal and nuclear alterations adeptly. The investigation, encompassing 100 female patients with a mean age of 47.5 years, elucidates the demographic and clinicopathological parameters. Predominantly presenting as palpable lumps (85%), invasive ductal carcinoma emerged as the predominant histological type (98%). The primary focus of the study revolves around the morphological changes post-neoadjuvant chemotherapy, with a meticulous qualitative analysis encompassing stromal elements (fibrosis, elastosis, calcification) and nuclear features (pyknosis, hyperchromasia). Notably, the response to chemotherapy, classified by the International Union against Cancer criteria, delineates a substantial pathological complete response (55%), partial response (35%), and limited non-response (10%). The therapeutic landscape includes a majority of cases undergoing extensive chemotherapy cycles, primarily featuring the cyclophosphamide, doxorubicin, and paclitaxel regimen. Remarkably, this investigation unveils fibrosis (63%) and elastosis/collagenization (51%) as prevalent stromal changes, while pyknosis (58%) and hyperchromasia (48%) dominate nuclear alterations. In conclusion, this retrospective study provides a comprehensive overview of post-neoadjuvant chemotherapy breast cancer specimens, shedding light on the intricate interplay of clinical parameters, treatment responses, and histopathological changes. The findings underscore the pivotal role of pathologists in accurately diagnosing and grading tumors in the evolving landscape of breast cancer management.

Temporal exposure to chronic unpredictable stress induces precocious neurobehavioral deficits by distorting neuromorphology and glutathione biosynthesis in zebrafish brain.

Modelling of chronic stress conditions in experimental animals and its neuropsychiatric outcomes has been well documented in literature. Zebrafish (Danio rerio) by exhibiting significant genetic and epidemiological similarities with human beings, has now emerged as a promising animal model of translational research. In this line, risk assessment following exposure to chronic unpredictable stress (CUS) towards neurobehavioral response and neuromorphology of sensitive brain region in zebrafish is the prime objective of the present study. With the existing knowledge on CUS in affecting diverse neurobehavioral aspects, we were primarily interested in whether this neurobehavioral transformation is an outcome of altered glutathione biosynthesis in zebrafish. We were also concerned about whether the precocious neurobehavioral transformation has been linked to altered neuromorphology in the periventricular grey zone (PGZ) of the zebrafish brain. Our basic findings showed that CUS itself represented as a universal factor in altering native bottom-dwelling and scototaxis behaviour of zebrafish. Our findings also backing the argument that CUS itself represented a collective stress regimen by altering the brain glutathione biosynthesis in zebrafish. Correspondingly, a temporal transformation in CUS instigated augmentation in neuronal pyknosis and chromatin condensation were observed in PGZ of the zebrafish brain. Collectively, these findings designate that CUS induced temporal neurobehavioral transformation is an outcome of augmented oxidative stress and neuromorphological alteration in the zebrafish brain. However, the underlying mechanism of such neuropathological manifestation associated with CUS might provide novel insight towards the development of prophylactic/therapeutic intervention to counter such co-morbid behavioral alteration.

Cell death in the male and female rat medial prefrontal cortex during early postnatal development.

Apoptosis, programmed cell death, is a critical component of neurodevelopment occurring in temporal, spatial, and at times, sex-specific, patterns across the cortex during the early postnatal period. During this time, the brain is particularly susceptible to environmental influences that are often used in animal models of neurodevelopmental disorders. In the present study, the timing of peak cell death was assessed by the presence of pyknotic cells in the male and female rat medial prefrontal cortex (mPFC), a cortical region that in humans, is often involved in developmental disorders. One male and one female rat per litter were sacrificed at the following ages: postnatal day (P)2, 4, 6, 8, 10, 12, 14, 16, 18, and 25. The mPFC was Nissl-stained, the densities of pyknotic cells and live neurons were stereologically collected, and the number of pyknotic cells per 100 live neurons, pyknotic cell density, and neuron density were analyzed. Males and females showed a significant peak in the ratio of pyknotic to live neurons on P8, and in females, this elevation persisted through P12. Likewise, the density of pyknotic cells peaked on P8 in both sexes and persisted through P12 in females. The timing of cell death within the rat mPFC will inform study design in experiments that employ early environmental manipulations that might disrupt this process.

Exposure of Radiofrequency Electromagnetic Radiation on Biochemical and Pathological Alterations.

INTRODUCTION: In the era of globalization, too much dependency on mobile phones is a cause of concern. OBJECTIVE: The present study was designed to evaluate the risk assessment of microwave radiation (MWR) at 1800 MHz frequency and specific absorption rate 0.433 (W/kg) on male Wistar rats. METHODOLOGY: Animals were divided into two groups: the first group is the control group, and the second group was exposed to 1800 MHz radiation for 90 days at 4 h/5 days/week in a month. RESULTS: Chronic exposure of MWR may alter GSH homeostasis due to alteration in various GSH cycle regulating enzymes such as GR, GPx, GST, and G6PDH which showed an imbalance in GSH content and causes an increase in the oxidative stress and release of inflammatory cytokines. A remarkable increase in the DNA damage was seen due to disorganization and pyknosis of neurons in exposed animal's brain when compared with the control group (P ≤ 0.05). There was also a significant decline in AChE level. CONCLUSION: The study concludes that MWR may cause neurochemical and pathophysiological damage by initiating the inflammatory process in various brain regions, especially in hippocampus and cerebral cortex. These effects are further associated with a remarkable elevation in the genotoxicity of neurons with reference to the control group.

The Genotoxic and Cytotoxic Effects of CT Scan on Buccal Epithelial Cells.

BACKGROUND: Diagnostic radiation is reported to cause significant damage in buccal cells, while the same effects after natural cell turn over cycle were not checked for in previous studies. The buccal cells were studied in patients exposed to computed tomography (CT) scans for evaluating the cells with micronuclei and cytotoxic changes, namely, pyknotic cells, karyorrhectic cells and karyolytic cells. The pre-exposure counts were compared with postexposure counts on 10 and 20 days corresponding to first and second cell turnover cycles. AIM: The aim of this study is to estimate the counts of micronucleus and cytotoxic changes in buccal cells post-exposure to CT scans and report on variance of the same with first and second buccal cell turnover cycles. MATERIALS AND METHODS: This is an observational study, wherein the buccal smears of patients undergoing CT scans were made before and after CT scan exposures as needed. Papanicolaou (PAP) staining and analysis were performed as per standard criteria for micronuclear and cytotoxic changes, respectively. Statistical test used was paired t-tests. RESULTS: The micronuclear counts revealed 0.4% positive cells before exposure and 1.4% positive cells post 10 days and 20 days of exposure were significant (P < 0.005). The cytotoxic changes showed around 2.5% positive cells before and 5.7% positive cells 10 days after CT exposure (P < 0.005). The cytotoxic cell values from baseline to 20th day were not significant (P < 0.25). CONCLUSION: CT scans have caused genotoxic effects notable after two cell turnover cycles but the cytotoxic changes have significantly decreased naturally after 2nd cell turnover as per our study.

Distribution and sequence of pyknotic cells in rat fetuses exposed to busulfan.

Busulfan, an antineoplastic bifunctional-alkylating agent, is known to induce developmental anomalies. In the present study, we examined the distribution and sequence of pyknotic cells in rat fetal tissues exposed to busulfan. Pregnant rats on gestation day 13 were administered intraperitoneally 30 mg/kg of busulfan, and fetal tissues were examined at 6, 12, 24, 36, 48, 72 and 96 hours after treatment (HAT). Pyknosis of component cells was observed markedly in the brain, moderately in the eyes and spinal cord and mildly in the craniofacial tissue, mandible, limb buds, tail bud, ganglions, alimentary tract, lungs, kidneys, pancreas and liver. In the brain, mitotic inhibition was also detected. Most of the pyknotic cells were considered to be apoptotic cells judging from the results of TUNEL staining and electron microscopic examination. Commonly in the above-mentioned tissues, pyknotic cells began to increase at 24 HAT, peaked at 36 or 48 HAT and disappeared at 96 HAT, which is when the histological picture returned to normal in most tissues except for the brain, spinal cord and eyes. The present study clarified the outline of busulfan-induced apoptosis in rat fetuses.

DNA damage assessment with buccal micronucleus cytome assay in Turkish coal miners.

The aim of this study was to assess DNA damage in Turkish coal miners with the buccal micronucleus cytome (BMCyt assay as the least invasive and therefore most practical method that may find wider application in coal miner biomonitoring. Buccal epithelial cell samples were taken from 54 coal miners and 42 controls from Zonguldak, Turkey to establish their micronucleus (MN), binucleus (BN), condensed chromatin (CC), karyorrhectic (KHC), karyolytic (KYL), nuclear bud (NBUD), and pyknotic (PYC) frequencies. We also analysed the effects of confounding factors such as age, years of work at the mine, smoking, alcohol drinking, and use of protective equipment on differences in MN frequencies. Two miners had confirmed and three suspect pneumoconiosis, whereas 49 displayed normal chest radiographs. MN, BN, KHC, and NBUD frequencies were significantly higher in coal miners than controls. Years of work at the mine also showed a significant effect on buccal MN frequencies in coal miners, but we found no correlation between MN frequencies and age, smoking, and alcohol consumption. In conclusion, BMCyt assay proved itself an accurate and practical screening method, as it can detect DNA damage much earlier than pneumoconiosis develops.