Bridging basic science and applied diagnostics: Comprehensive viral diagnostics enabled by graphene-based electronic biosensor technology advancements.

This study presents a graphene field-effect transistor (gFET) biosensor with dual detection capabilities for SARS-CoV-2: one RNA detection assay to confirm viral positivity and the other for nucleocapsid (N-)protein detection as a proxy for infectiousness of the patient. This technology can be rapidly adapted to emerging infectious diseases, making an essential tool to contain future pandemics. To detect viral RNA, the highly conserved E-gene of the virus was targeted, allowing for the determination of SARS-CoV-2 presence or absence using nasopharyngeal swab samples. For N-protein detection, specific antibodies were used. Tested on 213 clinical nasopharyngeal samples, the gFET biosensor showed good correlation with RT-PCR cycle threshold values, proving its high sensitivity in detecting SARS-CoV-2 RNA. Specificity was confirmed using 21 pre-pandemic samples positive for other respiratory viruses. The gFET biosensor had a limit of detection (LOD) for N-protein of 0.9 pM, establishing a foundation for the development of a sensitive tool for monitoring active viral infection. Results of gFET based N-protein detection corresponded to the results of virus culture in all 16 available clinical samples and thus it also proved its capability to serve as a proxy for infectivity. Overall, these findings support the potential of the gFET biosensor as a point-of-care device for rapid diagnosis of SARS-CoV-2 infection and indirect assessment of infectiousness in patients, providing additional information for clinical and public health decision-making.

Nuclear AgNOR protein enhancement in nucleoplasms of peripheral blood lymphocytes of babies/children with Down syndrome.

Down syndrome (DS) is one of the most common chromosomal disorders. The factors contributing to the mental retardation together with other defects in this syndrome have not been fully explained. Individuals with DS have extra rRNA gene family since they carry an extra chromosome 21. The few reports available are on the relationship between the nucleolus organizer regions (NORs) and DS phenotype. The in vivo regulation of NORs expression on the extra chromosome 21 is not completely understood. Previous studies have shown that nucleoli of lymphocytes from infants (mostly neonates) with DS contain more in vivo and in vitro nucleolar AgNOR proteins when compared with healthy infants. The objective of this study is to compare the in vivo nuclear AgNOR protein level in nucleoplasms (also called as karyoplasm) of nonstimulated peripheral blood lymphocytes from babies/children with DS and healthy controls. Peripheral blood samples obtained from 20 babies/children with DS and 20 matched healthy controls were smeared on clean glass slides and then AgNOR staining was performed. The AgNOR protein level in nucleoplasms of lymphocytes from both groups was calculated using a computer program. Nearly 100 interphase nuclei per individual were analysed. Average nuclear AgNOR protein levels in nucleoplasms of lymphocytes from babies/children with DS were found to be significantly higher than those of the controls (P < 0.001). On the basis of our present results, we propose that the increase of nuclear AgNOR protein in in vivo conditions may contribute to the formation of DS phenotypes.

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

BACKGROUND: 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. AIM: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

AgNOR status in Down's syndrome infants and a plausible phenotype formation hypothesis.

Down's syndrome (DS) or trisomy 21 is the most frequent genetic birth defect associated with mental retardation. Although DS has been known for more than a 100 years and its chromosomal basis recognized for half a century (1959), the underlying patho-mechanisms for the phenotype formation remain elusive and cannot be fully explained by simple gene dosage effect. The general consensus is that the extra chromosome 21 genes perturb the global metabolism of the body cells. Our experiments show that the most prominent metabolic perturbation occurs during ribosome biogenesis in the cells of DS babies/infants. In humans, ribosomal RNA (rRNA) gene families or nucleolar organizer regions (NORs) are localized at the secondary constriction (on the satellite stalks) of five pairs of acrocentric chromosomes (13, 14, 15, 21 and 22) and their activities are evaluated specifically either in metaphase or interphase through a procedure known as AgNOR or silver staining. Our successive AgNOR studies, supported by RNA and nuclear protein measurement, show that cells from DS infants produce more ribosomes than expected, accounting for the extra set of active rRNA gene family (1/6-1/11) situated on the extra chromosome 21. Thus, the presence of an extra chromosome 21 stimulates a global increase in ribosome biogenesis in cooperation with other NOR-bearing chromosomes, causing unnecessary rRNA and ribosomal proteins synthesis compared to controls. Following the description of NORs, AgNOR, AgNOR-proteins, AgNOR measurement and our experimental results, we propose that the extra RNA and protein synthesis can cause a fundamental handicap to DS infants, contributing to the formation of DS phenotypes, due to the wasted energy in producing unnecessary macromolecules, including energy (GTP)-dependent transport of the excessive ribosomes from the nucleus to the cytoplasm.

Toxic-dose warfarin-induced apoptosis and its enhancement by gamma ionizing radiation in leukemia K562 and HL-60 cells is not mediated by induction of oxidative stress.

The purpose of this study was to test the hypothesis that warfarin may enhance free radical production and oxidative damage on cancer cells. We examined the possible concentration-dependent effect of warfarin on cytotoxicity with respect to oxidative stress on leukemia cell lines (K562 and HL-60) and normal human peripheral blood mononuclear cells (PBMC). Gamma radiation was used as a positive control agent for oxidative stress. At all concentrations of warfarin (5-200 muM), 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol)- and bis-N-methylacridinium nitrate (lucigenin)-amplified chemiluminescence responses and lipid peroxidation and protein oxidation were stable after 72 h incubation at 37 degrees C. However, The 2',7'-dichlorofluorescein diacetate (DCFH-DA) oxidation was increased when cells were incubated with high concentrations (50-200 muM) of warfarin. In these concentration ranges, warfarin reduced cell growth in a dose-dependent manner, producing apoptosis. Our results also revealed that at concentrations above 5 muM, warfarin had a potentiating effect on radiation-mediated growth inhibition and apoptosis. Furthermore, marked effects were observed on leukemic cells compared with PBMC. We report here that the increase of DCFH oxidation might be due to the increase in the release of cytochrome C caused by warfarin, as cytosolic cytochrome C content was significantly elevated in the warfarin-treated cells compared with control cells, and because cotreatment with antioxidants N- acetylcysteine or 4,5-dihydroxy-1,3-benzene-disulfonic acid (Tiron) was unable to prevent cytochrome C release and DCFH oxidation induced by the drug. Taken together, these results suggest that high warfarin concentrations may be toxic to leukemic cells in vitro through apoptosis, although at the pharmacological concentrations (<50 muM), warfarin has no prooxidant or cytotoxic effect on PBMC, K562, and HL-60 cells. In addition, when the treatment of leukemic cells with warfarin at concentrations above 5 muM is combined with radiation, we observed an increase in radiation-induced cytotoxicity. The mechanism by which warfarin potentiates this cytotoxicity is unclear, but it may not be directly due to toxic damage induced by warfarin-generated free radicals.

AgNOR increase in buccal epithelial cells of trisomy 21 infants.

The aim of this study is to compare the Argilophilic Nucleolus Organizer Regions (AgNORs) level between Down syndrome (DS) patients and controls in a tissue sharing the same embryonic origin with the central nervous system and compare the results with those obtained recently by us from DS's lymphocytes. For this, buccal desquamating epithelial cells well known as the ectodermic origin were used. Since the AgNOR staining intensity is an indicator of the ribosomes biosynthesis rate, comparison of the image analysis values of the AgNOR area/total nuclear area (NORa/TNa) in buccal desquamating epithelial cells of DS patients and controls provided a plausible conclusion about the regulation/deregulation of the rRNA genes (rDNA) in these cells of DS babies/infants. The (NORa/TNa) proportion was calculated using an in-house computer program. Fifty buccal desquamating cells were analysed for each individual to determine the average NORa/TNa value per individual. In contrast to healthy controls, NORa/TNa proportion value of buccal epithelial cells from DS patients found significantly higher than that of the controls: (4.08+/-1.16)% and (2.13+/-0.55)%, respectively. This 92% increase is far higher than the expected value due to the extra rRNA genes on the extra-chromosome 21. Finally DS babies/infants exhibit very higher AgNOR expression increase in their buccal epithelial cells compared to controls. This is the first study that is available on the comparison of AgNOR expression levels in buccal epithelial cells between DS infants and their controls.

Nuclear protein contents in peripheral blood mononuclear cells of trisomy 21 infants.

OBJECTIVE: The trisomy 21 (Ts21) or Down's syndrome (DS) phenotype is assumed to occur primarily by the expression/overexpression of some genes encoded by the extra chromosome 21. It has recently been shown by AgNOR staining that babies with Ts21 have more AgNOR area (more NOR proteins) and more RNA content in their peripheral blood mononuclear cells (PBMCs) than those of controls. The aim of this study was to test whether or not the nuclear proteins content of PBMCs from trisomy 21 babies/infants is higher than that of the controls. METHOD: For this purpose, flow cytometric measurement of the stained PBMC nuclei was used. Nuclei from PBMCs was isolated and stained with propidium iodide and fluorescein isothiocyanate (PI/FITC) for DNA and protein estimation, respectively. RESULTS: Mean nuclear protein content of Ts21's (N = 30, mean age = 3.46 +/- 3.05 years old) PBMCs was found statistically higher than that of the controls (N = 33, mean age = 3.79 +/- 1.93 years old) (P = 0.005, nonparametric Mann-Whitney U test for two independent variables). This means that the average nuclear protein content of PBMC from Ts21 infants is higher than that of the controls. Furthermore, there is a moderate negative correlation between the ages of the studied DS patients and the protein content in the nuclei of their PBMCs (Linear regression analysis: P = 0.002, r = -0.55). This correlation is not found with controls (P = 0.186, r = -0.24). CONCLUSION: We have concluded that average protein content of PBMCs' nuclei from DS infants is higher than that of the controls, decreasing significantly with age.

Investigation of micronucleus frequencies in lymphocytes of inhabitants environmentally exposed to chrysotile asbestos.

Exposure to asbestos minerals has been associated with a wide variety of adverse health effects including lung cancer, pleural mesothelioma, and cancer of other organs. Many of the regions of Turkey have asbestos deposits. People in Doganli village - one of these regions - have been environmentally exposed to chrysotile asbestos since they were born. In this study the effects of asbestos on micronucleus (MN) frequencies of inhabitants exposed to chrysotile asbestos have been examined. Thirty subjects who had been environmentally exposed to chrysotile asbestos and living in Doganli village, and 25 controls were studied to assess the MN frequency. The control group was selected from healthy individuals with no exposure to asbestos and living in similar geographic conditions to Doganli village. Peripheral blood samples were collected from each subject and cultured for MN assay. Cytochalasin-B was added to lymphocyte cultures for evaluation of MN in binucleated (BN) cells. The differences between those exposed to chrysotile asbestos and controls were not statistically significant in terms of BN cells with MN (p > 0.05). There was not a significant relationship between MN frequencies and age, sex, smoking, both in chrysotile asbestos-exposed subjects and in controls (p > 0.05). Although the detection of calcified pleural plaques found in the inhabitants has indicated environmental exposure to chrysotile asbestos, our results show that chrysotile asbestos was not an inducer of MN in subjects exposed to chrysotile asbestos.

Age-dependent decreases in mitogen-stimulation level and RNA content in peripheral blood mononuclear cells of Down syndrome patients.

OBJECTIVE: The aim of the present study was to determine whether or not the phytohemagglutinin (PHA)-activated proliferation and average RNA content in peripheral blood mononuclear cells (PBMC) of Down syndrome (DS) patients change with age. METHOD: Stimulated portion of PBMC and total RNA levels in these cells after 72 h of PHA stimulation from 38 DS patients were compared with 28 age-matched healthy controls using flow cytometric measurement. RESULTS: Decreased ratio of PBMC from DS patients undergoes mitogenic stimulation with age (r = -0.84, P = 0.000). This decrease is not observed in the cells of control individuals (r = 0.03, P = 0.869). Stimulated PBMC in infants with DS have higher level of RNA contents compared to controls (Z = 2.227, P = 0.026). While RNA content in mitogen-stimulated PBMC of DS decreased progressively and significantly with age (r = -0.70, P = 0.000), no significant age-related change in RNA content was found among the cells of healthy individuals in the range of 0-27 year old (r = 0.275, P = 0.157, P > 0.05). CONCLUSION: Age-dependent decreases in mitogen-activated proliferation ratio and average RNA content of PBMC from DS patients appear as regular events. These results may contribute to the explanation of the immune deficiency seen in DS patients since the PHA-stimulated cells are principally T-lymphocytes. This is the first report on the decrease in PHA-stimulated proliferation ratio (stimulability) and RNA level in PBMC of DS patients in relation to age.

Two-dimensional agnor evaluation as a prognostic variable in urinary bladder carcinoma: a different approach via total agnor area/nucleus area per cell.

Traditional criterions are not sufficient to predict accurately the recurrence of transitional cell carcinoma of the urinary bladder. Therefore, we aimed to evaluate the AgNORs via total AgNOR area/nucleus area (TAA/NA) for each cell as a prognostic parameter, in TCC of urinary bladder. Tumor tissues of 20 consecutive cases of male bladder cancer patients were divided into two groups as middle differentiated (LG) and high grade (HG). The extra-tumoral tissue (ETT) samples of 10 males served as control group. A second control group (HC) consisted of five healthy and normal bladder tissue samples. The 3 microm of sections from each paraffin embedded tumoral, extra-tumoral and normal tissue samples served as patient and control groups. After deparaffinization and rehydratation steps, silver (AgNO(3)) staining of nucleolar organizer regions-associated proteins (AgNORs) was performed. Instead of Giemsa stain, we used Hematoxylin for contra staining. The images of the 100 analyzable nuclei from each tissue sample, transferred by means of a video camera and video capture card from microscope and recorded onto a computer. Software was prepared in Delphi language for analysis. Mean (E+02) TAA/NA values of HC, ETT, LG and HG groups were 6.97+2.80, 5.70+1.82, 7.80+3.22 and 9.24+3.88, respectively. Statistical comparisons have shown significant differences between all groups. In conclusion, mean TAA/NA per cell has a potential to be a prognostic parameter. Therefore, further evaluation of big patient series will be useful.

Increased micronucleus frequency after oral administration of cadmium in dogs.

Cadmium (Cd) is a toxic heavy metal that has been classified as a human carcinogen by the International Agency for Research on Cancer. The genotoxic effects of cadmium oxide (CdO) were investigated in cultured dog lymphocytes after a short-term oral CdO administration by the micronucleus (MN) test. The dogs were given 10 mg CdO/kg body weight per day for 3 and 28 d, respectively group I (n = 7) and group II (n = 6). Blood samples were collected at the beginning of feeding and at 4 and 29 d after Cd administration and cultured for 72 h. Whereas no significant increase in the MN frequency in group I was observed (p = 0.398), a significant MN induction with CdO was found in group II (p = 0.028) when compared with initial MN frequencies of dogs in both groups. Our results suggest that CdO might be directly and/or indirectly genotoxic after a monthly oral administration of CdO in dogs.

Flow cytometric comparison of RNA content in peripheral blood mononuclear cells of Down syndrome patients and control individuals.

OBJECTIVE: Trisomy 21 or Down syndrome (DS) is the most common genetic cause of mental retardation associated with the immunologic and other known defects. Extra chromosome 21 of DS patients contains an average of 40 extra copies of rRNA genes and the in vivo regulation of these genes' activity is not known. Because over 80% of total cellular RNA is rRNA, the measurement of total cellular RNA provides information on rRNA content. The aim of this work was to determine whether or not the additional chromosome 21 causes any increase in total cellular RNA content in mononuclear cells from peripheral blood (PBMNCs) of these patients and whether or not this content is modified with age. METHOD: PBMNCs of 48 patients with DS and 48 healthy controls were studied. RNA content of isolated PBMNCs was evaluated by flow cytometric measurements. RESULTS: Average RNA content of younger DS patients' cells was significantly higher than that of healthy controls (P=0.003). Furthermore, the RNA content decreased significantly with increasing age of DS patients (r=-0.377, P=0.008) in the range of 0-26 year old, whereas no significant relationship was found between age and PBMNCs' RNA content of healthy controls in the same range of ages. CONCLUSION: RNA content of PBMNCs from DS patients decreases rapidly with age. This is the first work on the age-dependent decrease of the RNA content in PBMNCs of DS patients.

NOR expression increases in interphase lymphocytes of Down syndrome babies/children as AgNORs surface, according to the mitogen concentration in the culture medium.

The extra chromosome 21 of Down syndrome (DS) or trisomy 21 patients contains an average of 40 extra copies of rRNA genes and the in vivo/in vitro regulation of the activity of these genes is not fully understood. The objective of this work was to compare the NORs expression pattern in interphase lymphocytes of DS patients with regular trisomy 21 and control individuals according to phytohemagglutinin (PHA) concentration (0.37, 0.75, 1.48 and 2.21 ml) per 100 ml of medium. Because the AgNOR staining is an indicator of the active rRNA genes, comparison of the image analysis values of the AgNOR area in 72 h cultivated lymphocytes for each concentration of PHA between DS patients (N=30) and controls (N=24) provided a plausible conclusion on the regulation of the extra rRNA genes in DS lymphocytes. The nucleolus organizer regions area/total nuclear area (NORa/TNa) was calculated using an in-house computer program. Fifty consecutive interphases per PHA concentration were analysed for each individual, for determination of the NORa/TNa. In contrast to healthy controls, NORa/TNa of lymphocytes from DS patient babies/children (0-8 years old) increased gradually in parallel with the PHA concentration in the culture medium: 10.44+/-1.72% for 0.37 ml of PHA, 11.74+/-1.93% for 0.75 ml of PHA, 13.25+/-2.03% for 1.48 ml of PHA and 13.43+/-2.08% for 2.21 ml of PHA per 100 ml of medium. Contrary to control cells (in which the NORa/TNa ratio according to PHA concentration in the culture medium remains constant), DS interphase lymphocytes in culture do not down-regulate their NOR expression. These results obtained from interphase NORs are consistent with the previous results obtained by evaluating the mean of AgNOR+ chromosome number in metaphase cells, also in relation to the mitogen concentration in the culture medium.

Evaluation of the nucleolar organizer regions in Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disorder in middle and late age. Ribosomal RNA (rRNA) genes are located in the nucleolus (nucleolar organizer regions = NORs). There are increased deposits of beta-amyloid protein in the brains of the patients with AD and aged individuals with Down's syndrome (DS). The beta-amyloid gene is located in the acrocentric chromosome 21 that is responsible for rRNA synthesis. Therefore, it is possible that there is a relationship between ribosomal genes and AD. OBJECTIVE: To investigate the activities of ribosomal genes of AD patients by comparing the activities of NORs in AD patients and healthy controls with the silver-staining method. METHODS: NOR surface/the total nucleus surface proportions in interphase nuclei, and silver stainability and satellite association (SA) of acrocentric chromosomes in the metaphases of cultivated lymphocytes of 20 AD patients and 20 healthy controls (10 elderly and 10 young) were evaluated. RESULTS: A decrease in NOR surface/total nucleus surface proportions has been observed in the interphase nucleus of AD patients when compared with elderly controls (p = 0.035). When compared with the sizes of Ag+ segments of acrocentric chromosomes of AD patients and control groups, the Ag-staining size 1 of the chromosome 22 of AD patients was found to be more increased than that of the young controls (p = 0.018). There was no statistically significant difference between AD patients and control groups regarding the number of Ag+ acrocentric chromosomes, Ag+ chromosome 21 and SA frequency (p > 0.05). It has been found that there is only a slight increase in the total number of chromosomes in SA in AD patients when compared with elderly controls (p = 0.05). CONCLUSION: The decrease in NOR surface/total nucleus surface proportions of AD patients may indicate a reduction in the activity of the ribosomal genes of these patients.

Higher NORs-expression in lymphocyte of trisomy 21 babies/children: in vivo evaluation.

Extra chromosome 21 of Down syndrome (DS) or trisomy 21 patients contains an average of 40 extra copies of rRNA genes and the in vivo regulation of these genes is not known. The objective of this work is to compare the NORs expression in interphase nuclei in non-stimulated lymphocytes of DS patients and healthy controls. Because the AgNOR staining is the indicator of the active rRNA genes, comparison of the image analysis values of AgNORs area between DS's and healthy controls' interphase lymphocytes is considered to be sufficient to evaluate the level of rDNA activities in the two groups. The Nucleolus Organizer Regions area/Total Nuclear area (NORa/TNa) was calculated using a computer program designed by us. 100 consecutive NORa/TNa per individual were evaluated. We report that 24 DS children's peripheral lymphocytes show significantly higher NORa/TNa mean value (6.32 +/- 1.77%) than that of the 20 healthy controls' cells (5.31 +/- 1.34%) (2-tailed Mann-Whitney U test, z = 19.4, P = 0.000). The same is true for the nucleolus (AgNOR spot) number per nucleus. The mean value of nucleoli number per nucleus in DS lymphocytes was significantly higher than that of the controls: z = 14.6, P = 0.000. In conclusion, extra rRNA genes on the chromosome 21 are not down-regulated in DS patients' lymphocytes. Rather, extra NORs expressions in 'in vivo' condition contribute to the increase of AgNORs area and AgNOR spots number per nucleus. This is the first work on the comparison of NORs activities in resting (non-stimulated) interphase lymphocytes between DS and healthy controls.

NOR expression increases on metaphase chromosomes of Down syndrome lymphocytes in concordance with mitogen concentration in culture medium.

BACKGROUND: Regulation of nucleolus organizer region (NOR) expression in trisomy 21 (Down syndrome [DS]) cells is not fully explained. This work compared NOR expression on metaphase chromosomes in gradiently stimulated lymphocytes from DS patients with those from healthy controls. METHOD: Conventional peripheral blood culture (72 h) and chromosomal preparation procedures were used except that blood samples from each individual were cultivated in the same but gradiently increasing concentrations (0.37, 0.75, 1.48, and 2.21 ml) of phytohemagglutinin (PHA) per 100 ml of medium. One hundred consecutive metaphases per concentration were analyzed for scoring the means of the active NORs bearing chromosomes (AgNOR+ chromosome) per individual and per concentration. RESULTS: In contrast to healthy controls (n=24), AgNOR+ chromosomal number in lymphocytes from 30 DS patients increased in concordance to the gradient of PHA concentration in the culture medium. CONCLUSION: DS lymphocytes do not downregulate their NOR expression in the limit of control cells. This in vitro result may serve as a clue for the explanation of the DS phenotype due to the wasted energy in producing unnecessary rRNA transcripts and AgNOR proteins in utero during organogenesis. These results also indicate that precautions must be used in routine work of NOR evaluation/interpretation in DS lymphocytes.

Micronucleus evaluation in mitogen-stimulated lymphocytes of PUVA treated patients.

PUVA describes the treatment of patients with psoralens plus an exposure to a source of UV light of 320-400 nm (UVA). Contradictory results have been reported on the chromosomal damage of PUVA when assayed by sister chromatid exchange (SCE) method. Micronucleus (MN) test is used to detect both clastogenic (breaking) and aneugenic (abnormal segregation) effect of physical/chemical agents on the chromosomes. No data have been found on the MN formation in the cells of PUVA treated patients. Frequency of micronuclei in 72 hours cultivated/mitogen-stimulated lymphocytes of patients have been evaluated at zero time and after 20, 40, 60 sessions of PUVA treatment. While the beginning MN frequency was approximately 0.22% (n=23), it raised to approximately 0.32 (n=23), approximately 0.42 (n=14) and approximately 0.53% (n=10) corresponding respectively to 20, 40 and 60 sessions. These sessions correspond reciprocally to 54+/-23, 172+/-48, 300+/-61 joules/cm2 of UVA and 13, 26, 39 mg/kg of 8-metoxypsoralen (8-MOP). While large interindividual variances were apparent, highly significant differences have been observed between initial MN frequency and after that of the 20, 40 and 60 sessions, (p = 0.000, p = 0.004, p = 0.005, reciprocally, Wilcoxon two-related samples test). The coefficient of correlation between MN frequency and UVA doses starting from zero to 60 sessions of treatment has been found as r = 0.61. This indicates a significant relationship between UVA doses and MN frequencies. However, MN inducibility and synergistic property of 8-MOP with UVA should be taken into account. Gradual MN increase during different sessions of PUVA treatment shows that--once appeared--a part of MN at least persist in the cells of patients from a few days to a few weeks. Smoking as a confounding factor seems to increase MN frequency (p = 0.053, Mann-Whitney U-test) in the beginning population, taken as the control population. This is the first report on the kinetics of MN formation during different sessions of PUVA treatment. Based on our results, we concluded that PUVA treatment causes a detectable chromosome damaging effect on the relatively profound cells/tissues of its human users. Therapists should be careful with its use, especially on the patients who may be more susceptible to carcinogenesis (e.g. immunosuppressed and/or elderly subjects).