ABSTRACT
Objective Blood trace and extraction is the important premising for forensic medicine appraisal reliability, this paper puts forward the blood trace and extraction method of using pyramidon. Methods Using pyramidon and benzidine to treat diluted fresh blood, and using Chelex-100 reagent treatment after the DNA in blood samples, and then applying the technology of PCR-STR and fluorescence detection test sample STR typing, observing the STR classification results and the detection sensitivity of DNA, comparing the different influence of two method on subsequent DNA - STR inspection. Results The pyramidon method can detect the minimum hemoglobin concentration is 25μg/mL,which was lower than 5μg/mL of the benzidine method. The results showed that the effective typing rate of the benzidine method was 18.09%, which was significantly lower than 96.67% of the method of the pyramidon method. The tail length of the detection group was 52.40±9.21, and the Oliva tail moment was 43.29±4.85%, and the tail intensity was 16.25±2.35, which was significantly higher than that of the pyramidon group (P < 0.01). Conclusions The sensitivity of the pyramidon method is moderate, and the influence of the following STR subtype and the nuclear DNA of the sample is significantly lower than that of benzidine, which can be used in the pre-experiment of blood stain in forensic medicine.
ABSTRACT
Objective To explore the effect of benzidine test and related reagents on DNA analysis of bloodstain. Methods A total of 970 bloodstain filter paper samples with 1μL venous blood were collected, and 10 of them acted as control samples. After benzidine test and related reagent processing, DNA of 960 samples was extracted by Chelex-100 and silica bead methods and then multiplex amplified by AmpF(e)STRTM IdentifilerTM Plus PCR kits. The results of STR typing were compared between different groups. Results DNA were extracted immediately after benzidine test. Totally STR loci (3.80±1.34) were detected by silica bead method, while no STR loci were obtained by Chelex-100 method. Thirteen sam-ples (21.7％) with whole STR typing results were obtained by drying after benzidine test, and the STR locus number (12.90±1.49) which obtained by silica bead method was much higher than by Chelex-100 method (4.70±1.96) (P<0.05). When DNA was extracted immediately after the addition of glacial acetic acid, the STR locus number was (9.40±2.09) by silica bead method, but no STR typing result was obtained by Chelex-100 method. All 15 STR loci could be obtained by only adding glacial acetic acid after drying and only adding tetramethylbenzidine alcoholization liquid or 3％ hydrogen peroxide liquid. Conclusion Benzidine test has significant influence on DNA analysis of bloodstain. The Chelex-100 method is not suitable for the DNA extraction of bloodstain after benzidine test. Drying after benzidine test and silica bead methods can effectively enhance the STR locus number of bloodstain.
ABSTRACT
Objective To develop a sensitive and stable substrate for enzyme‐linked immunosorbent assay(ELISA)and carry out methodology evaluation tests on it .Methods A kind of substrate with tetramethyl benzidine and urea peroxide as its main compo‐nents were made up ,its sensitivity ,precision and storage stability were evaluated and compared with other three kinds of substrate . Results This substrate was more sensitive than two of the three commercial substrates .The variable coefficient of precision test was 3 .5% .The optical densities(OD) of the new substrate after stored one day ,seven days ,one month ,six months ,twelve months in 4 ℃ environment were 2 .268 、2 .403 、2 .358 、2 .278 、2 .330 respectively ,the standard deviation was 0 .056 185 ,the variable coeffi‐cient was 2 .4% .Conclusion The substrate proposed in this paper displays good sensitivity ,precision and stability .The preparation method is simple ,reproducible .This substrate not only satisfies the requirements of laboratory research ,but also meets with the de‐mands for commercial kit development due to its function as an important component of test kits .It is an economical and effective choice for both laboratories and research transformation .
ABSTRACT
The effects of polymerization conditions including scan potential range, scan cycles, the concentration ratio of template molecules to functional monomer, pH of the buffer, and washing time for removing the template molecule from the imprinted polymer on the difference of zero current potential of benzidine ( BZ) interaction with BZ-MIP were investigated. The optimum preparations were obtained. The imprinted capacity of benzidine, 4-chloroaniline, and 4-aminobiphenyl and carmine was calculated as 0. 632, 0. 1123, 0. 1123, 0. 0847 and 0. 0725, respectively. This indicated that BZ-MIP had good specific recognition and selectivity to benzidine, and other substances did not interfere with the binding of BZ-MIP with BZ. The zero current potential variation was linear with the lorgarithm of BZ concentration in the range of 4í10-8-1í10-5 mol/Lwith detection limitation of 1. 89í10-8 mol/L. The sensor was used to detect BZ in waste water sample with recoveries of 95 . 7%-104 . 2%.
ABSTRACT
OBJECTIVES: To evaluate the effects on the formation of benzidine-hemoglobin, and benzidine metabolite-hemoglobin adducts, caused by pretreatment with the known xenobiotic metabolism effectors, ethanol and phenobarbital, in rats administered Direct Black 38 dye. METHODS: The experimental rats were divided into three groups: a control group, an ethanol group and a phenobarbital group. Rats were pretreated with ethanol (1g/kg) or phenobarbital (80mg/kg) 24 hours prior to the oral administration of Direct Black 38 (0.5mmol/kg), with the control group being administered the same amount of distilled water. Blood samples were obtained from the vena cava of 5 rats from each group prior to, and at 30 min, 3 h, 6 h, 9 h, 12 h, 24 h, 48 h, 72 h, 96 h, and 144 h following the oral administration of Direct Black 38. Directly after sampling the blood was separated into hemoglobin and plasma, with the adducts being converted into aromatic amines by basic hydrolysis. Hydrolyzed benzidiene, monoacetylbenzidine and 4-aminobiphenyl were analyzed by reverse-phase liquid chromatography with an electrochemical detector. The quantitative amount of the metabolites was expressed by the hemoglobin binding index (HBI). RESULTS: In the ethanol group, benzidine-, monoacetylben-zidine-, and 4-aminobiphenyl-HBI were increased to a greater extent than those in the control group. These results were attributed to the ethanol inducing N-hydroxylation, which is related to the formation of the hemoglobin adduct. In the phenobarbital group, all the HBIs, with the exception of the benzidine-HBI, were increased to a greater extent than those of the control group. These results were attributed to the phenobarbital inducing N-hydroxylation related to the formation of the hemoglobin adduct. The N-acetylation ratio was only increased with the phenobarbital pretreatment due to the lower benzidine-HBI of the phenobarbital group compared to those of the control and ethanol groups. The N-acetylation ratios for all groups were higher than 1 for the duration of the experimental period. Although the azo reduction was unaffected by the ethanol, it was inhibited by the phenobarbital. The ratio of the benzidine-HBI in the phenobarbital group was lower than those of the ethanol the control groups for the entire experiment. CONCLUSION: Our results indicate that both ethanol and phenobarbital increase the formation of adducts by the induction of N-hydroxylation, but also induced N-acetylation. Phenobarbital decreased the formation of benzidine-HBI due to the decrease of the azo reduction. These results suggest that the effects of ethanol and phenobarbital need to be considered in the biochemical monitoring of Direct Black 38.
Subject(s)
Animals , Rats , Administration, Oral , Amines , Chromatography, Reverse-Phase , Ethanol , Hydrolysis , Metabolism , Phenobarbital , Plasma , WaterABSTRACT
OBJECTIVES: The effects of ethanol and phenobarbital,which are known to affect metabolism of xenobiotics, on the formation of benzidine-and its metabolites-plasma protein adducts in rats administered benzidine were evaluated. METHODS: The experimental rats were divided into the control,ethanol and phenobar-bital groups. The experimental groups (ethanol and phenobarbital group)were pretreated with ethanol (1g/kg)or phenobarbital (80mg/kg)24 hours prior to the oral administration of benzidine (0.5mmol/kg). Blood samples were obtained from the vena cava from 5 rats in each group; and at 30 min,3 h,6 h,9 h,12 h,24 h,48 h,72 h,96 h,and 144 h after the administration of benzidine using heparin treated syringes.The plasma protein levels were separated immediately after taking blood samples. The adducts were underwent basic hydrolysis to convert them into aromatic amines. The hydrolyzed benzidine, monoacetylbenzidine, and 4-aminobiphenyl were analyzed by reverse-phased liquid chro-matography with an electrochemical detector. The quantitative amount of the metabolites was expressed by the plasma protein binding index(PBI). RESULTS: Similar to the hemoglobin adducts,the levels of the plasma protein adducts of the ethanol and phenobarbital groups (benzidine-, monoacetylbenzidine-, and 4-amino-biphenyl-PBI)were higher than those of the control group. These results are attributable to the fact that ethanol and phenobarbital induced to the plasma protein adduct formation. The N-acetylation ratio in the control group was highest at 72 h with 2.34.In the ethanol group,it was highest at 72 h with a ratio of 2.46 and was highest in the phenobarbital group at 72 h with a ratio of 2.43. The N-acetylation ratio of the plasma protein adducts was relatively lower than that of the hemoglobin adducts.The level of the plasma protein adduct increased more rapidly than the hemoglobin adducts in all experimental groups regardless of the pretreatment,and decreased rapidly after reaching the maximum level. CONCLUSION: The above results indicate that ethanol and phenobarbital increased the level of plasma protein adduct formation. The plasma protein adducts tended to decrease more rapidly than the hemoglobin adducts in the body after benzidine exposure. This results in this study result suggests that the effects of ethanol or phenobarbital need to be considered in the biochemical monitoring,and that the level of the plasma protein adducts be a more proper biomarker than the hemoglobin adducts for assessing the short term exposure to a benzidine and benzidine based dye.
Subject(s)
Animals , Rats , Administration, Oral , Amines , Environmental Monitoring , Ethanol , Heparin , Hydrolysis , Metabolism , Phenobarbital , Plasma , Protein Binding , XenobioticsABSTRACT
This is the first report on an occupational bladder cancer in Korea. The ease is 41 years old man who worked as a dyer for 17 years at two dyeing factories, which handled nylon and polyester fabrics in Taegu. He was exposed to many kinds of dyes during weighing, mixing, dissolving and dyeing processes. Among many kinds of acid, disperse and direct dyes that he has been exposed to, several dyes have confirmed to contain benzidine-based dyes, one was o-tolidine-based dye, and one was o-dianisidine-based dye. He visited a hospital due to the gross hematuria and urinary frequency in June, 1998, and he had radical cystectomy with ileal conduit diversion. He had smoked a half-pack of cigarette for 20 years. The main risk factor of bladder cancer is smoking, however, he was relatively a light smoker than usual Korean men. He was exposed to the definite occupational carcinogen even though the level was relatively lower than that of dye manufacturers. His age was younger than the prevalence age of bladder cancer caused by smoking. These evidences support that the dyer' s bladder cancer could be related to the occupational exposure to benzidine-based dyes.
Subject(s)
Adult , Humans , Male , Coloring Agents , Cystectomy , Hematuria , Korea , Nylons , Occupational Exposure , Polyesters , Prevalence , Risk Factors , Smoke , Smoking , Tobacco Products , Urinary Bladder Neoplasms , Urinary Bladder , Urinary DiversionABSTRACT
This study is performed to evaluate usefulness of dermal measurement of benzidine and benzidine based dye as one of the occupational exposure assessment method for these compounds. We selected one benzidine manufacturing factory and one dye manufacturing factory in Incheon area. Eleven workers were for benzidine manufacturing factory and twenty four for dye. We analyzed relationships among air level, amount on skin and concentration of urinary metabolites for these compounds. Airborne levels of benzidine and dye were measured by NIOSH 5509, 5013 methods. Amount of these compounds on skin was measured with skin wipe method. Concentration of benzidine metabolites in urine was measured by High Performance Liquid Chromatography after alkaline hydrolysis. The amount of benzidine on hand skin was 25.05( - 233.2) ng/ur, and the amount of the neck was 2.01 ( - 11.9) ng/cm2 in the benzidine dihydrochloride manufacturing factory. The amount of benzidine on hand and neck skin has positive correlation with concentration of urinary monoacetyl benzidine (r=0.644, p < 0.05) . The amount of benzidine based dye on hand skin was 55.75( - 457.7) ng/cm2, and the amount of the neck skin was 18( - 284.7) ng/cm in benzidine based dye manufacturing factory The amount of dye on hand and neck skin has positive correlation with concentration of urinary benzidine for dye workers (r=0.467, p < 0.05). When assessing the exposure of workers who deal with benzidine, the amount of benzidine on skin should be measured for an accurate exposure assessment.
Subject(s)
Chromatography, Liquid , Hand , Hydrolysis , Neck , Occupational Exposure , SkinABSTRACT
Direct Black 38, a kind of benzidine-based azo dye, is widely used as a dye for fabric, leather, cotton, cellulosic material, paper, wool, silk, and so on. Benzidine-based azo dyes are proven as a mutagen and linked to bladder cancer. In 1978, Natonal Institute for Occupational Safety and Health recommended that three widely used benzidine-based dyes (Direct Black 38, Direct Blue 6, and Direct Brown 95) should be treated as carcinogens. In this experiment, metabolism of the benzidine-based dye. Direct Black 38 was examined by using an isolated liver perfusion system. To measure the metbolites of Direct Black 38,/ 8.0 micrometer, 30.5 micrometer and 63,3 micrometer of Direct Black 38 was added into the recirculating perfusate of the isolated perfused rat liver. Samples were collected at 0, 10, 20, 30, 60, 90. 120 minute. They were treated with sep-pak and methanol, and the metabohtes were detected and quantified with high performance liquid chromatography (HPLC). Residual non-reactive dye in the perfusate and liver was reduced to benzidine and then analyzed by HPLC. Detected metabolites of ?Direct
Subject(s)
Animals , Rats , Carcinogens , Chromatography, High Pressure Liquid , Chromatography, Liquid , Coloring Agents , Liver , Metabolism , Methanol , Occupational Health , Perfusion , Silk , Urinary Bladder Neoplasms , WoolABSTRACT
To evaluate,the differences of benzidine exposure patterns of the workers in two benzidine-based dye manufacturing factories, the concentration of benzidine: in. air, blood, and urine were measured. The air levels of benzidine dihydrochloride and benzidine-based dye were measured by high performance liquid chromatography with ultraviolet detector. Blood samples were collected at 3 hours after exposure and urine samples were collected at the end of shift. Blood and urine samples were analyzed by high performance liquid chromatography with electrochemical detector. The level of benzidine in reaction process (input, diazotization, and coupling); was 0.381+/-7950 g/m3. The blood benzidine was deteced in 25 workers among 38 in reaction process and their mean levels were 0.0153?0376 ng/mg Hb. The urinary benzidine was detected for 11 workers among 38 workers in the reaction process. The level of benzidine-based dye in drying and packing process was 52.1748+/-4.4111g/m3. The blood benzidine was deteced in 6 workers among 38 in drying and packing process and their mean levels was 0.0062+/-0274 ng/mg Hb. The urinary benzidine was detected for 1 worker among 38 workers exposed to benzidine-based dye. The blood and urinary benzidine were detected in workers exposed to benzidine-based dye. Such results suggested that some part of benzidine-based dye was metaboized to benzidine. Therefore, some regulations for manufacturing and use of the benzidine-based dye are needed to prevent its hazards in industries.
Subject(s)
Chromatography, Liquid , Social Control, FormalABSTRACT
Benzidine Industry in Korea has started after Japan has banned its production in early 1970's. and it has been in operation in Korea for over 20 years. However, it is not known yet whether any bladder cancer has developed from benzidine exposure. This study was done to screen benzidine-exposed workers for bladder cancer, and to examine the feasibility of employing screening test at the workplace. All the workplaces that manufacture or use benzidine for more than 20 years in Korea have been covered in this study, and they include 2 benzidine manufacturing factories, 5 benzidine using factories, as well as 2 benzidine free factories as an outside control. In total, 516 workers were screened with urine stick test and urine cytology test for the evidence of hematuria and abnormal urothelial cells. Each worker was also asked about risk factors and symptoms of bladder cancer including past medical history, smoking, medication and occupational history. Benzidine in the air was measured by personal and area sampling. Out of 516 screened workers, 84(16.3%)workers showed positive hematuria in urine stick test, and 7(1.4%)workers showed degenerative cells in urine cytology tests. Those workers with abnormal urine test results who have been exposed to benzidine for more than 10 years were further screened, and, in total. 23 workers were examined with intra-venous pyelography and cystoscopy. None of those screened had any evidence of bladder cancer. When workers with only past hematuria history were included in the positive hematuria group, 96(18.5%) had positive hematuria. On the multiple logistic regression analysis, positive hematuria was significantly associated with benzidine exposure history of other occupations with elevated bladder cancer risk, pyuria and glycosuria. The association got stronger as direct benzidine exposure was accounted through individual task analysis, and as exposure duration was accounted with tenure analysis. For those with benzidine exposure with more than 10 years of tenure, the odds of having positive hematuria was elevated 2.14(95%C.I is 1.08 to 4.25) times more than for those without exposure. Even though bladder cancer was not detected for several limitations including short observation period, majority of studied workers with short latency, healthy worker effect, and low sensitivity of single screening test in a cross-sectional study, the study results suggest that hematuria screening is a feasible and very useful test for bladder cancer screening among benzidine exposed workers.
Subject(s)
Humans , Cross-Sectional Studies , Cystoscopy , Glycosuria , Healthy Worker Effect , Hematuria , Japan , Korea , Logistic Models , Mass Screening , Occupations , Pyuria , Risk Factors , Smoke , Smoking , Urinary Bladder Neoplasms , UrographyABSTRACT
Potential toxicities of three classes of dyes and pigments were introduced in this review. Some azo/benzidine dyes, anthraquinone dyes and triarylmethane dyes have carcinogenicity,some azo/benzidine dyes and anthraquinone dyes may cause skin irritation, some noncarcinogenic triarylmethane dyes have risk of serious damage to eyes and may exert long term adverse effects on the aquatic environment.
ABSTRACT
DNA in the livers of rats was isolated 24 h af-ter oral administration of benzidine, Congo red andEvan's blue, DNA adducts in the livers of ratswere investigated by a ~(32)P-postlabeling assay. Thelevel of DNA adducts in the livers of rats after oraladministration of benzidine, congo red andEvan'blue were 18. 17, 1. 03 and 1. 74?mol/kgDNA respectively. The results showed that themetabolites of the benzidine derivative azo dyes canform adducts with DNA in the livers of rats.These DNA adducts probably are one of the rea-sons that the incidence of the bladder cancer rosein the people group who are only exposed to benzi-dine derivative azo dyes.
ABSTRACT
Objective To investigate the effects of hexamethylene bisacetamide(HMBA),3-(2-hydroxy-3-methyl-butyrylamino-)-propionic acid(HMBPA) and HMBA plus HMBPA on MELDS19 cell growth and differentiation.Methods To make the MELDS19 cell growth curve,count the MELDS19 cell mitosis index,numbers of colony forming and benziding staining positivity percentage when MELDS19 cells exposed to HMBA,HMBPA and HMBA plus HMBPA.Results The numbers of the MELDS19 cells exposed to 2mmol/L HMBA was 5.0?10~5/ml,to 1?mol/L HMBPA was 6.0?10~5/ml and to 1?mol/L HMBPA+2mmol/L HMBA was 3.3?10~5/ml respectively,mitosis index and colong forming decreased.Benzidine staining positivity percentage exposed to 2mmol/L HMBA was 38%,to 1?mol/L HMBPA was 9% and to 1?mol/L HMBPA+2mmol/L HMBA was 64% respectively.Conclusion HMBA and HMBPA can inhibit MELDS19 cell proliferation and induce differentiation much effectively than HMBA or HMBPA used alone.