Exfoliated Bladder Cells Research Articles

EMERGING ROLE OF URINARY BIOMARKERS IN DETECTION OF UROTHELIAL BLADDER CARCINOMA IN SOUTH INDIAN POPULATION

Urine cytology is used for screening of exfoliated bladder cells from voided urine but lacks sensitivity. This study aims to check the efficacy of 5-aminolevulinic acid (5- ALA) fluorescence cytology and establish a high sensitivity approach in detecting flat, in-situ and/or small lesions that are hardly visible under conventional cystoscopy. Intracellular PPIX allows red fluorescence detection. In this study, 5-ALA fluorescent cytology using urine was compared with conventional cytology in the diagnosis of bladder tumors. In this prospective study, we compared the sensitivity and specificity between conventional cytology, 5-ALA fluorescent cytology and FDA approved commercially available kits (NMP-22 and BTA). The percentage of Protoporphyrin IX facilitated by 5-ALA was amplified in cancer urothelial cells compared to normal urothelial cells. The sensitivity of conventional cytology and 5-ALA fluorescent cytology was 64% and 96% respectively, whereas the specificity was 92% and 98.67% respectively. In conclusion, 5-ALA induced fluorescent urine cytology demonstrated promising outcomes in the detection of bladder carcinoma cells. Furthermore, low grade and low stage tumor cells as well as flat lesions were also positively and accurately interpreted using 5-ALA fluorescent cytology.

Assessment of deoxyribonucleic acid damage in exfoliated bladder cells and its prognostic implication in urinary bladder cancer patients

Background: In this era where when one has to look for prognostic and diagnostic methods for evaluating tumors not only they are invasive but very much expensive also which is not at all easy for everyone to opt for. In our study we have tried to look for Deoxyribonucleic Acid (DNA) damage in exfoliated bladder cells which is obtained from a simple urine test and tried to correlate the damage with tumour stage and grade obtained from bladder biopsy. Aim: To assess Deoxyribonucleic Acid (DNA) damage in Bladder tumour and it’s extrapolation on exfoliated bladder cells and to study association of DNA damage markers with diseases prognosis if any. Study and Design: It is analytical cross sectional study done in tertiary care centre in Kolkata, West Bengal, India. Material and Method: After fulfilling inclusion and exclusion criterion we evaluated 70 patients with Bladder space occupying lesion (SOL) to study the DNA damage on exfoliated bladder cells by Fast Halo method and Gamma Histone Analysis and studied the correlation between them if any and also Histopathological Findings. Statistical Analysis Used: All statistical tests were done using GraphPad PRISM (version 7, 2016) and SPSS 20 for Windows ( IL, USA). Association was determined by Student t-test and correlation study was performed by Spearman’s correlation coefficient. Results: It was concluded that DNA damage assessed by Fast Halo method done on exfoliated bladder cells statistically correlates with the findings of DNA damage assessed by Gamma Histone analysis and there was a positive correlation with tumour grade and stage. Conclusion: DNA damage assessed by Fast Halo method correlates well with the finding of DNA damage assessed by Gamma Histone analysis and both these findings were consistent with the findings obtained by Histopathology.

Association between chromosomal aberration of exfoliated bladder cells in the urine and oxidative stress in patients with bladder transitional cell carcinoma.

The aim of the current study was to investigate the chromosomal aberrations of exfoliated bladder cells in the urine and blood oxidative stress in patients with bladder transitional cell carcinoma (BTCC). A total of 40 healthy controls and 246 patients with BTCC were recruited. Abnormal levels of CSP3, CSP7, CSP17 and GLPp16 were detected by fluorescence in situ hybridization (FISH) in exfoliated bladder cells in the urine of patients with BTCC. Serum total oxidant status (TOS), total antioxidant status (TAS) and oxidative stress index (OSI) were measured. Significant differences were observed in the abnormal CSP3, CSP7, CSP17, GLPp16 signals and FISH positive rate between patients with BTCC and healthy controls (P<0.001). Serum TOS, TAS and OSI were also significantly different between the two groups (P<0.001). The clinical stage of BTCC was not associated with abnormal CSP3, CSP7, CSP17, GLPp16 or FISH positive rate and oxidative stress (P>0.05). A Gamma rank correlation analysis revealed an association between the pathological grade of BTCC with abnormal CSP3, CSP7 and CSP17 as well as FISH positive rate (P<0.001). In addition, the clinical stage of BTCC was associated with serum TOS, TAS and OSI (P<0.001). Evaluation of the association between chromosomal aberrations and oxidative stress revealed that abnormal CSP3, CSP7 and CSP17 were positively associated with serum TOS and OSI (P<0.001), abnormal CSP7 and CSP17 were negatively associated with serum TAS (P<0.001), but abnormal GLPp16 was not associated with serum TOS, TAS or OSI (P>0.05). Therefore, the chromosomal aberrations of exfoliated bladder cells in the urine are associated with blood oxidative stress in patients with BTCC, and these factors may contribute to the occurrence and development of BTCC.

Effect of Increased Water Intake on Urinary DNA Adduct Levels and Mutagenicity in Smokers: A Randomized Study.

The association between fluid intake and bladder cancer risk remains controversial. Very little is known about to which extent the amount of water intake influences the action of excreting toxics upon the urinary system. This proof of concept trial investigates the effect of water intake on mutagenesis in smokers, a high risk population for bladder cancer. Methods. Monocentric randomized controlled trial. Inclusion Criteria. Male subjects aged 2045–45 y/o, smokers, and small drinkers (24-hour urinary volume <1 L and osmolality >700 mOsmol/kg). Outcomes. 4-ABP DNA adducts formation in exfoliated bladder cells in 24-hour urine collection and urinary mutagenicity in 24-hour urine. Test Group. Subjects consumed 1.5 L daily of the study product (EVIAN) on top of their usual water intake for 50 days. Control Group. Subjects continued their usual lifestyle habits. Results. 65 subjects were randomized. Mean age was 30 y/o and mean cigarettes per day were 20. A slight decrease in adducts formation was observed between baseline and last visit but no statistically significant difference was demonstrated between the groups. Urinary mutagenicity significantly decreased. The study shows that increasing water intake decreases urinary mutagenicity. It is not confirmed by urinary adducts formation. Further research would be necessary.

Does radio frequency radiation induce micronuclei frequency in exfoliated bladder cells of diabetic rats?

For many years there has been a discussion among both experts and the general public regarding the effects of radio frequency (RF) radiation on the human organism. The purpose of the present study was to evaluate the relationship of micronucleui (MN) frequency and RF radiation in exfoliated bladder cells of non-diabetic and diabetic rats. Three groups were used in the experiment: Group I (n=6): diabetic group without RF exposure; Group II (n=6): diabetic group exposed 2100 MHz RF radiation and Group III (n=6): control animals (non-diabetic group, no RF exposure). RF exposure in the experiment resulted in a whole body average SAR of 0.24 W/kg with an ERMS field of 17.5 V/m in non-thermal levels. Results showed that there was no statistically important differences between non-RF exposed diabetes group and control group; Group I and Group III (p>0.05). There was no statistically important differences between diabetes group and diabetes+RF exposed group (Group I and Group II) (p>0.05). RF exposure did not result in increased MN frequencies in exfoliated bladder cells of diabetic rats with respect to control animals (Group II and Group III), either and this result found no statistically important (p>0.05). This study suggested no possible genotoxic effects of RF radiation among human beings especially with chronic disorders, such as diabetes.

Genetic Instability Persists in Non-Neoplastic Urothelial Cells from Patients with a History of Urothelial Cell Carcinoma

Bladder cancer is one of the most common genitourinary neoplasms in industrialized countries. Multifocality and high recurrence rates are prominent clinical features of this disease and contribute to its high morbidity. Therefore, more sensitive and less invasive techniques could help identify individuals with asymptomatic disease. In this context, we used the micronucleus assay to evaluate whether cytogenetic alterations could be used as biomarkers for monitoring patients with a history of urothelial cell carcinoma (UCC). We determined the frequency of micronucleated urothelial cells (MNC) in exfoliated bladder cells from 105 patients with (n = 52) or without (n = 53) a history of UCC, all of whom tested negative for neoplasia by cytopathological and histopathological analyses. MNC frequencies were increased in patients with a history of UCC (non-smoker and smoker/ex-smoker patients vs non-smoker and smoker/ex-smoker controls; p<0.001), in non-smoker UCC patients (vs non-smoker controls; p<0.01), and in smoker/ex-smoker controls (vs non-smoker controls; p<0.001). Patients with a history of recurrent disease also demonstrated a higher MNC frequency compared to patients with non-recurrent neoplasia. However, logistic regression using smoking habits, age and gender as confounding factors did not confirm MNC frequency as a marker for UCC recurrence. Fluorescent in situ hybridization analysis (using a pan-centromeric probe) showed that micronuclei (MN) arose mainly from clastogenic events regardless of UCC and/or smoking histories. In conclusion, our results confirm previous indications that subjects with a history of UCC harbor genetically unstable cells in the bladder urothelium. Furthermore, these results support using the micronucleus assay as an important tool for monitoring patients with a history of UCC and tumor recurrence.

No genotoxic effect in exfoliated bladder cells of rat under the exposure of 1800 and 2100 MHz radio frequency radiation

In this study, we aimed to investigate the effects of 1800 and 2100 MHz Radio Frequency (RF) radiation on the number of micronucleus (MN) in exfoliated bladder cells of rat which shows the genotoxic damage. Exposure period was 30 min/day, 6 days/week for a month and two months exposure periods. Thirty male wistar albino rats were used for five groups: Group I (n = 6): 1800 MHz RF exposed animals for one month, Group II (n = 6): 2100 MHz RF exposed animals for one month, Group III (n = 6): 2100 MHz RF exposed for two months, Group IV (n = 6): control group for one month, Group V (n = 6): control group for two months. Rats of the control groups were housed in their home cages during the entire experimental period without subjecting to any experimental manipulation. 1800 and 2100 MHz RF exposures did not result in any significant MN frequencies in rat bladder cells with respect to the control groups (p > 0.05). There was no statistically significant difference between 2100 MHz RF exposed groups, either. Further studies are needed to demonstrate if there is any genotoxic effect, micronucleus formation in other tissues of rats.

Arsenic trioxide: Pharmacokinetics in acute promyelocytic leukemia (APL) patients.

7055 Background: Arsenic (As) has significantly increased survival for APL patients. As and its metabolites’ effects on cell proliferation, apoptosis, and methylation, especially long term, remain largely unknown. It is imperative to study the pharmacokinetics of As at therapeutic doses in order to limit untoward effects resulting from treatment. Arsenic trioxide (ATO), available as arsenous acid (iAsIII), is readily metabolized through sequential methyl group additions and electron reduction steps: iAsIII →MAsV →MAsIII →DMAsV →DMAsIII →TMAsVO →TMAsIII. iAsIII, MAsIII and DMAsIII are more biologically active and more toxic than pentavalent forms. The key enzyme involved is arsenic methyltransferase, and polymorphisms contribute to metabolic differences between individuals. Methods: Cancer patients not treated with ATO (controls) had one collection of blood and urine samples, while APL patients receiving therapeutic doses of ATO had collections immediately prior to and at 1, 2, 4, 6, and 24 hours, days 4, 8, 15, and 4 weeks after ATO-free interval. Total iAs (iAsIII+iAsV), MAs (MAsIII+MAsV) and DMAs (DMAsIII+DMAsV) were measured in plasma using hydride generation cryotrapping atomic absorption spectrometry, a sensitive automated method of arsine detection. The same As species were measured in urine and in exfoliated bladder cells isolated from urine. Results: We report data on ten control patients and six treated patients (ATO 0.15 mg/kg/day). Initial average As concentrations in treated patients (0.051 ng/ml) were similar at baseline to the controls (0.046 ng/ml). We observed that iAs is quickly metabolized from a peak average plasma concentration of 32 ng As/ml to a trough of 10 ng As/ml within six hours from infusion, remaining unchanged for at least 24 hours. MAs and DMAs concentrations begin to increase at six hours, and continue to rise by day 4 to an average concentration of 8.5 and 10.4 ng As/ml respectively, followed by decline to baseline 4 weeks after final ATO infusion. Conclusions: Treatment with ATO leads to the formation of MAs and DMAs whose long term toxicity in APL patients is poorly understood. Studies involving analysis of As metabolites are needed to assess possible long term toxicities on non-targeted organs.

Is There Any Possible Genotoxic Effect in Exfoliated Bladder Cells of Rat Under the Exposure of 1800 MHz GSM-Like Modulated Radio Frequency Radiation (RFR)?

People are exposed to many carcinogenic and mutagenic chemicals in their everyday lives. These include antineoplastic drugs, Polycyclic aromatic hydrocarbons (PAH)s, aromatic amines, nitrosamines, metals, and electromagnetic radiation. Based on the state of knowledge acquired during the last 50 years of research on possible biological effects of electromagnetic fields (EMF), the majority of the scientific community is convinced that exposure to EMF below the existing security limits does not cause a risk to the health of the general public. However, this position is questioned by others, who are of the opinion that the available research data are contradictory or inconsistent and, therefore, unreliable.In this study, we aimed to investigate if there is any effect of 1800 MHz GSM modulated radio frequency radiation (RFR) on the number of micronucleus in exfoliated bladder cells of rat which will be informative about the genotoxic damage. Exposure period was 20 min/day, 5 days/week during a month. Six female Wistar rats were used for two groups: Group I (n=6): controls; Group II (n=6): 1.8 GHz exposed animals. 1800 MHz RFR did not showed a significant MN frequencies in rat bladder cells when compared with the control group (p>0.05). 1800 MHz RFR-exposed animals did not produce any genotoxic effect when compared with the control group (p>0.05). Kinetic studies are important for any biomarker, especially those in which tissue differentiation and maturation processes will heavily influence the time between induction of damage and collection of damaged cells for micronucleus analysis.

548 POSTER Micronuclei in exfoliated bladder cells of gynecological cancer patients receiving pelvic radiotherapy

548 POSTER Micronuclei in exfoliated bladder cells of gynecological cancer patients receiving pelvic radiotherapy

Randomized controlled trial: effects of diet on DNA damage in heavy smokers

We have conducted a randomized trial which investigated the ability of dietary changes (in particular diets rich in cruciferous vegetables and flavonoids), to increase urinary antimutagenicity and inhibit DNA damage in smokers. Ninety heavy smokers were recruited and randomly assigned to three groups, and were given three different diets. The first diet was based on flavonoid-rich foods, particularly cruciferous vegetables, but not based on supplementation; the second was a normal isocaloric diet (with an adequate administration of fruits and vegetables); and the third was based on supplementation of flavonoids in the form of green tea and soy products. DNA adducts were measured by (32)P-postlabelling in exfoliated bladder cells at different times since the start of the trial. In spite of randomization, subjects in the control group smoked more than those in the experimental groups, and this can explain the higher adduct levels at baseline. A slight decrease in bulky DNA adducts in exfoliated bladder cells was observed after 1 year since the end in the supplementation group and after 1 month in white blood cells. The only statistically significant association was found in a regression model that adjusted for smoking, in which the increase in flavonoid intake was associated with a decrease in adducts after 1 year (P = 0.02). These data suggest that adherence to a diet rich in cruciferous vegetables and flavonoids might reduce genotoxicity in the human urinary bladder of smokers, but they should be interpreted with caution owing to small numbers and the uneven distribution of smoking habits in the experimental groups. Smoking is the most important single preventable cause of cancer; at the present stage of knowledge it is totally unlikely that certain dietary habits can seriously counteract the effects of tobacco smoking.

Urothelial cell DNA adducts in rubber workers.

Workers employed in the rubber industry appear to have a significant excess cancer risk in a variety of sites, including cancer of the urinary bladder. In this cross-sectional study, we investigated the occurrence of DNA adducts in exfoliated bladder cells of currently exposed, nonsmoking rubber workers (n = 52) and their relationship with occupational exposure estimates and acetylation phenotype (NAT2). Four DNA adducts were identified, with the proportion of positive samples (e.g., DNA samples with quantifiable levels of a specific DNA adduct) ranging from 3.8 to 79%. The highest proportion of positive samples and the highest relative adduct labeling levels were in workers involved in the production functions "mixing" and "curing," areas with potential for substantial exposure to a wide range of chemical compounds used in rubber manufacturing (P < 0.05 for adducts 2 and/or 3, compared to all other departments). No statistically significant relationships were found between identified DNA adducts and urinary mutagenicity or personal inhalable and dermal exposure estimates. Interestingly, subjects with a fast NAT2 acetylation phenotype tended to have higher levels of DNA adducts. This study suggests that rubber workers engaged in mixing and curing may be exposed to compounds that can form DNA adducts in urothelial cells. Larger studies among rubber workers should be conducted to study in more detail the potential carcinogenicity of exposures encountered in these work areas.

Genes for human arylamine N-acetyltransferases in relation to loss of the short arm of chromosome 8 in bladder cancer.

Polymorphisms of N-acetyltransferase type 2 (NAT2) conferring the slow acetylator phenotype have been linked to increased susceptibility to arylamine-induced bladder cancer in Caucasians. Genes for NAT2, the other NAT isozyme, NAT1, and a NAT pseudogene (NATP) are found on 8p22, a region displaying loss of heterozygosity, particularly in invasive bladder tumours. A restriction enzyme digestion map has defined the relative positions of the NAT genes to each other and to adjacent CpG islands. NAT2, as a polymorphic gene of known function, is a potentially valuable marker for the detection of loss of heterozygosity in 8p22. Two approaches to investigate loss of heterozygosity at the NAT2 locus in bladder tumours have been used. (1) A cosmid containing NAT2 has been used in fluorescence in-situ hybridization on human exfoliated bladder cells collected from unselected bladder cancer outpatients. Loss of signal from the NAT2 cosmid was found in nine of the 20 patients. (2) A panel of 13 human bladder tumours was investigated for loss of heterozygosity using the polymorphism in the NAT2 gene as a marker. Loss of heterozygosity at the NAT2 locus has been compared with loss of heterozygosity at adjacent microsatellite marker sites known to be located on 8p. There is agreement between loss of heterozygosity at the NAT2 locus and adjacent microsatellite marker loci in 11 of the tumours but two of the tumours appear to show retention at the NAT2 locus. More extensive mapping of the region around the NAT loci, particularly on the centromeric side, is important to pinpoint possible tumour suppressor genes or their modifiers in the region. There are no other expressed sequences known in this region and therefore NAT genes are important genetic landmarks.

Modifying effect of vitamins C, E and beta-carotene against gamma-ray-induced DNA damage in mouse cells

The modifying effect of treatment with vitamins C, E and beta-carotene on the clastogenic activity of gamma rays was investigated in mice. Damage in vivo was measured by the micronucleus assay in bone marrow polychromatic erythrocytes and exfoliated bladder cells. The vitamins were administered orally, either for five consecutive days before or immediately after irradiation with 2 Gy of gamma rays. The results show that pretreatment with vitamin E (100–200 mg/kg/day) and beta-carotene (3–12 mg/kg/day) were effective in protecting against micronucleus induction by gamma rays. Vitamin C depending on its concentration enhanced the radiation effect (400 mg/kg/day), or reduced the number of micronucleated polychromatic erythrocytes (50–100 mg/kg/day). Such effect was weekly observed in exfoliated bladder cells. The most effective protection in both tissues was noted when a mixture of these vitamins was used as a pretreatment. Administration of the all antioxidant vitamins to mice immediately after irradiation was also effective in reducing the radiation-induced micronucleus frequency. The data from the in vitro experiments based on the comet assay show that the presence of the vitamins in culture medium influences the kinetic of repair of radiation-induced DNA damage in mouse leukocytes.

Relationship of urinary arsenic to intake estimates and a biomarker of effect, bladder cell micronuclei.

The purpose of this study was to investigate methods for ascertaining arsenic exposure for use in biomarker studies. Urinary arsenic concentration is considered a good measure of recent arsenic exposure and is commonly used to monitor exposure in environmental and occupational settings. However, measurements reflect exposure only in the last few days. To cover longer time periods exposure can be estimated using arsenic intake data, calculated by combining measures of environmental arsenic and inhalation/ingestion rates. We compared these different exposure assessment approaches in a population chronically exposed to arsenic in drinking water in northern Chile. The study group consisted of 232 people, some drinking water low in arsenic (15 micrograms/l) and others drinking water with high arsenic concentrations (up to 670 micrograms/l). First morning urine samples and questionnaire data, including fluid intake information, were collected from all participants. Exfoliated bladder cells were collected from male participants for the bladder cell micronuclei assay. Eight different indices of exposure were generated, six based on urinary arsenic (microgram As/l urine; microgram As/g creatinine; microgram InAs/l urine; microgram MMA/l urine; microgram DMA/l urine; microgram As/h, excreted), and two on fluid intake data (microgram As/day, ingested; microgram As/l fluid ingested-day). The relationship between the different exposure indices was explored using correlation analysis. In men, exposure indices were also related to a biomarker of effect, bladder cell micronuclei. While creatinine-adjusted urinary arsenic concentrations had the strongest correlations with the two intake estimates (r = 0.76, r = 0.81), unadjusted urinary arsenic showed the strongest relationship with bladder cell micronuclei. These data suggest that, in the case of the bladder, unadjusted urinary arsenic concentrations better reflect the effective target organ dose compared to other exposure measures for biomarker studies.

Tumor-Associated Antigens in Normal Mucosa of Patients With Superficial Transitional Cell Carcinoma of the Bladder

We identified premalignant lesions in tumor-surrounding histologically normal bladder mucosa from patients with superficial and muscle-invasive bladder cancer to look for "premalignant" lesions as the source of tumor recurrence. Tumor-associated antigen expression in histologically normal bladder mucosa in 23 patients with superficial bladder cancer was studied. Histologically normal bladder specimens from 21 patients with muscle-invasive bladder cancer and from 30 prostatic cancer patients served as controls. Tumor-related antigens 486p 3/12, LBS8, 19A211 and M344 were mapped quantitatively with monoclonal antibodies and immunohistological techniques. Expression of all four bladder tumor-associated antigens was significantly enhanced and more than two antigens were expressed simultaneously in histologically normal mucosa from both bladder-cancer groups, compared with histologically normal bladder mucosa of prostatic carcinoma patients (p < 0.05). The results suggest the existence of early malignantly transformed cells in the benign-looking and morphologically normal bladder mucosa of superficial bladder carcinoma patients, which possibly is the source of recurrent bladder cancer. This approach models the mapping of histopathologic premalignant lesions of the bladder to predict risk.

DNA adducts in urothelial cells: Relationship with biomarkers of exposure to arylamines and polycyclic aromatic hydrocarbons from tobacco smoke

Markers of exposure to polycyclic aromatic hydrocarbons (urinary 1-hydroxypyrene-glucuronide) and aromatic amines (4-aminobiphenyl-hemoglobin adducts), as well as urinary mutagenicity, were measured in 47 healthy smokers and 50 non-smokers. DNA adducts were determined by P32-postlabeling in the exfoliated bladder cells of 39 healthy subjects. Both 1-hydroxypyrene-glucuronide (1-OHPG) and 4-aminobiphenyl adducts (4-ABP-Hb) were associated with smoking habits, but only 4-ABP-Hb adducts were associated with consumption of black, air-cured tobacco. The levels of 2 DNA adducts (numbers 2 and 4) in urothelial cells were clearly associated with 4-ABP-Hb adducts, in all subjects and in smokers. Levels of one of these DNA adducts (number 2) were also associated with 1-hydroxypyrene-glucuronide in urines, but in smokers the association was not statistically significant. Overall, these observations constitute further evidence of a role of arylamines in tobacco-induced bladder cancer. © 1996 Wiley-Liss, Inc.

DNA adducts in urothelial cells: Relationship with biomarkers of exposure to arylamines and polycyclic aromatic hydrocarbons from tobacco smoke

Markers of exposure to polycyclic aromatic hydrocarbons (urinary 1-hydroxypyrene-glucuronide) and aromatic amines (4-aminobiphenyl-hemoglobin adducts), as well as urinary mutagenicity, were measured in 47 healthy smokers and 50 nonsmokers. DNA adducts were determined by P32-postlabeling in the exfoliated bladder cells of 39 healthy subjects. Both 1-hydroxypyrene-glucuronide (1-OHPG) and 4-aminobiphenyl adducts (4-ABP-Hb) were associated with smoking habits, but only 4-ABP-Hb adducts were associated with consumption of black, air-cured tobacco. The levels of 2 DNA adducts (numbers 2 and 4) in urothelial cells were clearly associated with 4-ABP-Hb adducts, in all subjects and in smokers. Levels of one of these DNA adducts (number 2) were also associated with 1-hydroxypyrene-glucuronide in urines, but in smokers the association was not statistically significant. Overall, these observations constitute further evidence of a role of arylamines in tobacco-induced bladder cancer.

Arylarnine N-acetyltransferase as a potential biornarker in bladder cancer: fluorescent in situ hybridization and irnmunohistochernistry studies

Abstract Arylamine N-acetyltransferase isoenzymes NAT1 and NAT2 are encoded at two polymorphic loci on human chromosome 8p22. The two loci have previously been identified using chimeric Yeast Artificial Chromosome (YAC) clones encoding either NAT1 or NAT2 as probes for metaphase chromosomes using fluorescent in situ hybridization. The 8p22 region has been demonstrated to be deleted in highly invasive bladder tumours and since NAT isoenzymes participate in the metabolism of arylamine bladder carcinogens, it is important to determine whether NAT1 and NAT2 gene loci are included in the region of deletion. We describe here the application of a cosmid clone for NAT2 as a biomarker for Fluorescent In Situ Hybridization (FISH) on interphase nuclei of exfoliated bladder cells. We also describe a 70kb probe for NAT1 which is a candidate for a suitable biomarker for use in similar FISH studies. lmmunohistochemical staining of bladder tumour sections with a polyclonal anti-peptide antibody specific for the NATl isoenzyme as a biomarker for NAT1 protein expression is also shown.

Use of the fluorescent micronucleus assay to detect the genotoxic effects of radiation and arsenic exposure in exfoliated human epithelial cells

The exfoliated cell micronucleus (MN) assay using fluorescent in situ hybridization (FISH) with a centromeric probe is a rapid method for determining the mechanism of MN formation in epithelial tissues exposed to carcinogenic agents. Here, we describe the use of this assay to detect the presence or absence of centromeric DNA in MN induced in vivo by radiation therapy and chronic arsenic (As) ingestion. We examined the buccal cells of an individual receiving 6,500 rads of photon radiation to the head and neck. Exfoliated cells were collected before, during, and after treatment. After radiation exposure a 16.6-fold increase in buccal cell MN frequency was seen. All induced MN were centromere negative (MN −) resulting from chromosome breakage. This finding is consistent with the clastogenic action of radiation and confirmed the reliability of the method. Three weeks post-therapy, MN frequencies returned to baseline. We also applied the assay to exfoliated bladder cells of 18 people chronically exposed to high levels of inorganic arsenic (In-As) in drinking water (average level, 1,312 μg As/L) and 18 matched controls (average level, 16 μg As/L). The combined increase in MN frequency was 1.8-fold (P = 0.001, Fisher's exact test). Frequencies of micronuclei containing acentric fragments (MN −) and those containing whole chromosomes (MN+) both increased (1.65-fold, P = 0.07, and 1.37-fold, P = 0.15, respectively), suggesting that arsenic may have both clastogenic and weak aneuploidogenic properties in vivo. After stratification on sex, the effect was stronger in male than in female bladder cells. In males the MN-frequency increased 2.06-fold (P = 0.07) while the frequency of MN+ increased 1.86-fold (P = 0.08). In addition, the frequencies of MN − and MN+ were positively associated with urinary arsenic and its metabolites. However, the association was stronger for micronuclei containing acentric fragments. By using FISH with centromeric probes, the mechanism of chemically induced genotoxicity can now be determined in epithelial tissues. © 1996 Wiley-Liss, Inc.