text: Chem. Listy 101, s73−s310 (2007) 12. mezioborová česko-slovenská toxikologická konference P46 PESTICIDE EXPOSURE ACCORDING TO THE CZECH TOXICOLOGICAL INFORMATION CENTRE FROM 1997 TO 2005 HANA RAKOVCOVÁa, DANIELA PELCLOVÁa, 39 % women; in 2 % the gender was unknown. During the BRONISLAVA ŘIČAŘOVÁa a TOMÁŠ NAVRÁTILb years, the percentage of these calls slowly decreased from 7.8 % in 1997 to 5.0 % in 2005. a Toxicological Information Centre, Department of Oc- Veterinary calls represented only 1.4 % of all calls, cupational Medicine, General University Hospital and but 12% of all calls due pesticides. The course of some Charles University in Prague, First Faculty of Medicine, intoxication was in life-threatening, with severe symptoms b J. Heyrovský Institute of Physical Chemistry ASCR (4.6 %) or lethal (5.0 %). v.v.i, Prague The exact numbers of inquiries in consecutive years email@example.com are given in Table I. Among insecticides, the decrease was seen especially in the group of organophosphates and car- bamates. More detailed data is shown in Table II. About Key words: Pesticides, Czech Toxicological Information 79 % of all exposures occurred in the vegetation period Centre, Rodenticides, Intoxication, Exposure, from April to October, and only 21 % calls in the other months. Introduction Pesticide poisoning was predominantly an accidental overdose. There were 91 % unintentional exposures, 6 % Pesticides became a part of our households and may suicidal, 2.3 % occupational and 0.7 % due to aggressive represent a danger, especially when they are ingested by behavior. Ingestion accounted for 84 %, inhalation for children or adults1,2. Some exposures lead to life- 13 % and skin contamination for 3 %. The symptoms of threatening intoxications3. However little has been pub- intoxication were absent in 50.5 % of subjects. About lished about the current situation and frequency of expo- 27.5 % of patients had mild symptoms, 8% medium, 1% sures to these substances in Central Europe. The aim of severe, in the rest of the cases the symptoms were not this study was to describe the development and severity of known at the time of the call. Death was the reason of exposures to pesticides, based on the calls to the Toxico- phone call in 0.3 % of cases. logical Information Centre, which serves to the Czech In exposures to organophosphates and carbamates, population of approximately 10 million. 47.4 % of the subjects had no symptoms; 27.4 % had mild symptoms, 8 % medium and 3 % severe, in other calls they Methods were unknown. Death was known in 1.5 % calls. Only 35 % calls concerned the children. Data taken from the Czech Toxicological Information In exposures to rodenticides 83 % patients were as- Centre database from periods 1997−2005 were evaluated ymptomatic, 5 % had mild symptoms, only 1.8 % medium retrospectively using electronic evidence system. and 2 % severe symptoms. In other calls the symptoms were not described. About 72 % calls involved children. Results In 2005, the hospitalization was found necessary only in 14.4 % of subjects. About 1/3 of them were sympto- Overall pesticide poisonings reached 4405 in this matic at the time of the call. Antidotal treatment was rec- period and accounted for 6.3 % of total calls to the Toxico- ommended in 1.6 % of calls only: one patient was given logical Information Centre. Forty-eight % calls concerned atropine, and the other phytomenadione. In 2005 only one adults, 40 % children, and 12 % animals. lethal case was registered. It concerned post-mortem con- In human exposures, 59 % of calls involved men, sultation due to suicide with pirimicarb. Table I Inquiries concerning types of pesticides to the Toxicological Information Centre in the years 1997−2005 1997 1998 1999 2000 2001 2002 2003 2004 2005 Insecticides 224 291 335 189 198 217 192 183 153 Rodenticides 102 140 105 142 136 150 105 118 164 Herbicides -- -- -- 83 109 117 78 77 89 Other* -- -- -- 110 122 191 117 95 73 Total 326 431 440 524 565 675 492 473 479 * Fungicides, moluscocides, combination or unknown pesticides. -- Data not available, included into Other s258 Chem. Listy 101, s73−s310 (2007) 12. mezioborová česko-slovenská toxikologická konference Table II Selected data from the inquiries concerning pesticides in the years 1997−2005 1997 1998 1999 2000 2001 2002 2003 2004 2005 Female, % 38.7 42.0 37.5 40.8 38.1 37.7 42.8 40.9 41.8 Accidental, % 82.4 85.6 91.3 88.0 93.3 92.4 89.2 92.0 88.3 Ingestion, % 81.7 86.0 82.5 83.8 85.8 85.2 82.1 81.5 86.0 Age 22.9 21.8 23.0 22.9 25.7 26.6 29.9 27.6 24.6 (mean, SD) (14.4) (20.4) (19.9) (14.4) (27.6) (30.9) (20.3) (24.6) (27.1) Time 15.5 (7.0) 16.4 (5.7) 14.7 (6.8) 13.9 (7.7) 14.1 (8.3) 14.6 14.1 (6.6) 14.1 (6.6) 14.5 (7.9) (mean, SD) (10.7) Month 6.9 (4.2) 6.5 (5.3) 6.3 (4.8) 6.8 (6.6) 6.6 (7.7) 6.6 (9.1) 6.4 (5.8) 6.4 (6.1) 6.4 (5.6) (mean, SD) Discussion Conclusions The development of inquiries to the Czech Toxico- Acute human pesticide exposure in our country is logical Information Centre concerning pesticides is rela- mainly accidental and has good prognosis in general, due tively favorable. It is true especially for human poisonings, to low toxicity of commercial products used. The number as the most dangerous products had already been replaced of calls to the Czech Toxicological Information Centre due by less toxic products. It can be seen in the group of or- to pesticides in the past 9 years is relatively stable and ganophosphates insecticides that have been substituted by shows a mild decrease since the year 2003. More impor- other substances, i.e., synthetic pyrethroids with low toxic- tant is the development of the spectrum of pesticides, in- ity for the mammals. The situation was even better in the gested by the patients either accidentally or intentionally. group of rodenticides. In the Czech Republic, solely war- During the past years, the number of calls concerning toxic farin or superwarfarin based rodenticides are commercially substances, such as organophosphates and carbamates available, which explains the good course of ingestions of insecticides slowly decreases. On the other hand, the num- these products. Comparing with the situation in the years ber of calls due to rodenticides mildly increased, however 1988−1989 (ref.1), the percentage of suicidal attempts with no serious sequel has been recorded. Lower number of pesticides from all exposures was not different from the deaths is the most important difference from the situation recent data. However, the severity of exposures substan- in the late 80ies, both after suicidal and non-intentional tially decreased. In the years 1988−1989, total 14 deaths ingestions. were recorded (7 after suicidal ingestions and 7 after drinking pesticide non-intentionally from a soft-drink bot- The study was supported by project tle). MSM0021620807. On the other hand, veterinary calls document a higher danger for exposed animals, which is in agreement with REFERENCES the literature4,5. One reason may be ingestion of a higher dose of the pesticide, when the animals are left alone with- 1. Pelclová D., Picková J., Filipová J.: Čas. Lék. Čes. out supervision. Another explanation is the repeated expo- 130, 361 (1991). sure, in some cases even intentional, i.e. by the neighbors, 2. Hruškovič I., Bátora I., Sagat T., Getlík A., Strnová J., which concerns especially rodenticides poisonings. In Milovský V., Zajíčkova M.: Česk. Pediatr. 39, 581 some instances improper use of antiparasitic preparations (1984). caused the poisoning of cats and dogs. 3. Ritter L., Goushleff N. C. I., Arbuckle T., Cole D., Raizenne M.: J. Toxicol. Env. Health 9, 441 (2006). 4. Vejlupková J., Fenclová Z.: Prakt. Lék. 80, 4 (2000). 5. Berny P.: J. Vet. Pharmacol. Ther. 30, 93 (2007). s259 Chem. Listy 101, s73−s310 (2007) 12. mezioborová česko-slovenská toxikologická konference P47 ENVIRONMENTAL RISK FACTORS OF ALLERGIC DISEASES IN 5-YEAR-OLD CHILDREN KATARÍNA RAUSOVÁa, ĽUBICA Table I PALKOVIČOVÁa, MONIKA URSÍNYOVÁa, Relation of risk factors and prevalence of allergic diseases VLASTA MAŠÁNOVÁa, EVA REICHRTOVÁa, among Slovak children PETER ČIŽNÁRb, LADISLAVA WSÓLOVÁa Risk Factors AD ARS RA AE FA a Research Base of the Slovak Medical University, Lim- Family history of AD ** ** * bová 14, 833 03 Bratislava, b Comenius University Medi- Maternal history of AD *** ** * cal Faculty, 1st Pediatric Department, Limbová 1, 833 40 Bratislava, Slovak Republic Paternal history of AD * * * firstname.lastname@example.com Region type ** * *** Type of delivery * Male gender ** * Key words: allergic diseases, children, environmental pol- Mother smoked in past * lution, risk factors, heavy metals Exclusive breast * * * P ≤ 0.05; ** P ≤ 0.01; *** P≤ 0.001 Introduction Rapid increase in the prevalence of allergic diseases tions of lead (median 26.5 µg l−1), cadmium (median (AD), which includes atopic eczema (AE), asthma respira- 0.128 µg l−1) and mercury (median 0.42 µg l−1) in 5-year- tory symptoms (ARS), rhinitis allergica (RA) and food old children's blood, we find a negative trend between the allergy (FA), in last decades and particularly in industrial- Cd levels in blood and incidence of AD in regions with ized regions is, besides genetic factors, ascribed to envi- different environmental characteristics (fig. 1). ronmental factors. Prevalence of allergic diseases, asthma respiratory symptoms and rhinitis allergica among children differed Subjects and methods with respect to environmental characteristics of the re- gions. Statistically significant differences were found in The following methods were applied: follow-up the frequency of allergic diseases (fig. 2) between regions: (years 1997−2003) of cohort of children (n=1997) from M/R and M/CH (P=0.01). Prevalence of asthma respira- birth to 5 years of age (n=403) for allergic disease devel- tory symptoms (fig. 3) differed between regions: CH/A opment in environmentally different Slovak regions, an- (P=0.05) and CH/M (P=0.01). Prevalence of allergic rhini- nual clinical examination of children by paediatric aller- tis (fig. 4) was different between regions: R/M (P=0.001), gists and administration of maternal questionnaires fo- R/CH, R/A and CH/M (P=0.01). cused on socio-economic status, life style and risk factors. Child's peripheral blood collection for analyses of toxic metals, analysis of Pb, Cd and Hg by atomic absorption spectrometry (AAS) method, statistical evaluation of asso- ciations in EpiInfo v.6 and SPSS v.12. 40 0,16 0,148 Four selected Slovak regions were selected according Prevalence of AD conc. of Cd [ug/L] 0,137 to different predominant environmental pollutants and 30 0,123 0,12 0,109 anthropogenic activities: industrial chemistry (CH) with 20 0,08 [%] the main sources of air pollution derived from chemical industry, industrial metallurgy and mining (M), agricul- 10 0,04 tural (A) and rural (R) without any point source of indus- trial pollution. 0 0,00 R CH A M Results Comparison (Chi Square and Mantel-Haenszel tests) of the prevalence of AD in 5-year-old children with rela- median Cd [ µg/L] tion to risk factors is given in Table I. Influence of parent's positive history of allergic diseases was confirmed. Al- Fig. 1. Prevalence of Allergic Diseases in 5-year-old children though we did not find significant influence of concentra- and Cd concentrations in blood s260 Chem. Listy 101, s73−s310 (2007) 12. mezioborová česko-slovenská toxikologická konference 40 40 Prevalence of RA [%] Prevalence of AD [%] ** ** 20 20 * *** * * 0 0 1 2 3 5 1 2 3 5 years years R CH A M R CH A M Fig. 4. Development of Rhinitis Allergica Fig. 2. Development of Allergic Diseases Conclusions Negative trend of AD prevalence with respect to Cd 40 blood levels support the knowledge of immunotoxic ef- Prevalence of ARS [%] fects of Cd. In our previous work, negative correlation was found between the concentrations of Cd and IgE levels in cord blood1. Differences in AD prevalence among regions 20 may result from different environmental exposures. * ** This work was supported by the Agency for U.S.- Slovak Science and Technology Program 012/95; Open 0 Society Foundation G 147/98; NPPZ 23 and Slovak Re- 1 2 3 5 search and Development Agency, APVT-21-025602. years CH R A M REFERENCE 1. Reichrtova E., Lang-Dunlop A., Palkovicova L., Fig. 3. Development of Asthma Respiratory Symptoms Ciznar P., Adamcakova A., Ursinyova M., Prachar, V., McNabb S. J. N.: ACI International 14, 117 (2002). s261 Chem. Listy 101, s73−s310 (2007) 12. mezioborová česko-slovenská toxikologická konference P48 HAZARD IDENTIFICATION OF BINARY CHEMICALS MIXTURES BY QSAR TECHNIQUES MARIAN RUCKI, MILOŇ TICHÝ, flow rate was 1.2 ml min−1. For analysis of samples gener- IVETA HANZLÍKOVÁ, RÚT UZLOVÁ ated in experiments was used isocratic mode 40 % A and 60 % B. The wavelength of detection was 254 nm. National Institute of Public Health, Šrobarova 48, 10042, Praha 10, Czech Republic D e t e r mi n a t i o n o f K m i x firstname.lastname@example.com Coefficient of distribution Kmix was determined ac- cording to4, using following equation: Introduction n ∑1+ W Qi QSAR and QAAR techniques applying knowledge of i =1 data on Tubifex tubifex test1,2 (EC50(T.t.)), R-analysis3 K1 = W × VKi mix n n (analysis of plot of mixed EC50(T.t.) against molar frac- V ∑Q − ∑ 1 + W Qi i tion of a binary mixture) and partition coefficient of com- i =1 i =1 pounds between n-octanol and water (log P) were used to VKi (1) identify additivity or non-additivity of acute toxicity in mixtures of chemicals and to study their nature. The where W is a volume of the aqueous phase, V is a volume Tubifex tubifex test takes 3 minutes exposure and was veri- of the octanol phase, n is a number of compounds in the fied by correlation with both log Kow and acute toxicity mixture, Qi is total amount of the compound i in the sys- indices measured with fish and ciliates1. tem and Ki is partition coefficient of compound i. The Materials and method value of Kmix is normalized, i.e. in case of additivity the resulting Kmix is equal 1. Following chemicals were used: 2-nitroanisole Scientific graphic and analyzing software ORIGIN® (Aldrich, 99.9+ %), phenol (Aldrich, 99.9+ %), n-octanol was used for statistical calculations. The additivity was (Aldrich, 99%), water (GORO AQUA 200, deionized, tested using chi-square test and t-test with Bonferonis cor- filtered through 0.22 mm membrane). rection. D e t e r mi n a t i o n o f K o w D e t e r mi n a t i o n o f E C 5 0 The studied compound is added to a system of n- The inhibition of the movement of oligochaeta octanol and water where the volume of both phases is ad- Tubifex was measured as the effective concentration that justed according to the expected value of Kow. For each causes 50 % of maximal response. Batches of six worms pair of tested compounds a series of binary mixtures with were immersed in aqueous solutions of the binary mixtures molar ratio of benzene 1.0, 0.9, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05, and the concentration – response curve was determined. and 0.0 was prepared. Each series was then tested at a sum The number of immobilized worms was counted in each concentration of both compound to be 0.005, 0.02 and batch precisely three minutes after their immersion. The 0.2 mol l−1. measurement was repeated three times with each concen- The system was shaken gently until equilibrium was tration on various days and in triplicate on each day. The achieved (5 to 10 hr) and then centrifuged to separate the reproducibility of the measurements was checked by the two phases, especially if an emulsion has formed. Both parallel determination of the EC50 for aqueous MnCl2 phases, i.e. the n-octanol and the aqueous phase, were solution1. directly analyzed for both studied compounds using gas chromatography or liquid chromatography. The GC sys- Results and discussion tem used consisted of Agilent 6890N gas chromatograph Acute toxicity EC50 (normalized value) of binary with Agilent 7683 series injector and FID detector. The mixture phenol-2-nitroanisole (fig. 1) is expressed as the column used was J&W scientific capillary column (30 m × inhibition of movement of oligochaeta Tubifex tubifex. The 0.25 mm × 0.25 m). Nitrogen (99.99 %) was used as dashed line shows EC50 in case of additivity (normalized a carrying gas. Injection volume was 1 ml. value 1). The normalization was used for purpose of a The HPLC system used consisted of two ECOM LCP better understandable explanation of toxic effect of mix- 4020 pumps, a Knauer dynamic mixing chamber, and an ture and for mathematical modeling. Values of normalized ECOM LCD 2083 UV/VIS detector. The column used was EC50 above dashed line indicate inhibition of the toxic a ChromolithTM Performance RP-18e (4.6 × 100 mm) with effect and values below dashed line indicate synergistic ChromolithTM Guard Cartrige RP-18e (4.6 × 5 mm) guard toxic effect. column (Merck, Prague, CR). Mobile phase for pump A Fig. 2 represented partition coeficient Kow of 2- consisted of degassed water and for pump B 4 : 1 metha- nitroanisole, fig. 3 partition coeficient Kow of phenol and nol/water mixture. Injection volume was 20 µl and the fig. 4 normalized coefficient of distribution Kmix of the s262 Chem. Listy 101, s73−s310 (2007) 12. mezioborová česko-slovenská toxikologická konference 2,0 32 1,8 30 1,6 Kow(Phenol) 28 EC50 (norm.) 1,4 26 1,2 24 1,0 0,8 0,0 0,2 0,4 0,6 0,8 1,0 RPhenol 0,0 0,2 0,4 0,6 0,8 1,0 Rnitroanisole Fig. 1. EC50 of the phenol-2-nitroanisole mixture Fig. 3. Kow of the phenol in the phenol-2-nitroanisole mix- ture 80 1,20 75 1,15 70 Kow (2-Nitroanisole) 1,10 65 1,05 Kmix(norm.) 60 1,00 0,95 55 0,90 50 0,85 45 0,80 40 0,0 0,2 0,4 0,6 0,8 1,0 0,0 0,2 0,4 0,6 0,8 1,0 RPhenol RPhenol Fig. 2. Kow of the 2-nitroanisole in the phenol-2-nitroanisole Fig. 4. Kmix of the phenol-2-nitroanisole mixture mixture mixture phenol-2-nitroanisole, total concentration of the The research was financially supported by Internal compounds were 0.02 mol l−1. Grant Agency of Ministry of Health of Czech Republic no. In the real life, the exposure to chemicals in mixture NR8780-3/2006, partly by European Union (European is more common than exposure to single compounds. Re- Commission, FP6 Contract no. 003956) and partly by sulting activity should be different from additivity ap- grant of Grant Agency of Czech Republic no. 203/06/1265 proach, widely used in studies on mixture toxicity5,6. The and by National Institute of Public Health. analysis indicates that the mixtures of industrial solvents phenol and 2-nitroanisole indicates clear "mixture interac- REFERENCES tion", i.e. inhibition or potentiation depending on ratio of components in the mixture. An attempt to simulate the relationship between EC50 and molar ratio of mixture with 1. Tichy M., Rucki M., Hanzlíková I., Roth Z.: ATLA log Kow is shown. 35, 229 (2007). Using of Kmix for QSAR modeling seems to be not 2. Tichý M., Rucki M.: Pracov. Lék. 48, 225 (1996) (in fully sufficient. As is shown at fig. 4, there are no big Czech). changes of the Kmix for whole range of the mixture. On the 3. Tichý M., Cikrt M., Roth Z., Rucki M.: SAR QSAR other hand, the log Kow of phenol and 2-nitroanisole show Environ. Res. 9, 155 (1998). significant differences from log Kow for both pure com- 4. Verhaar H. J. M., Busser F. J. M., Hermens J. L. M.: pounds. Especially the log Kow of 2-nitroanisole could Environ. Sci. Tech. 29, 726 (1995). explain the acute toxicity of phenol – 2-nitro-anisole. On 5. Hermens J. L. M., Canton H., Janssen P., de Jong R.: fig. 2 is shown decrease of Kow for 2-nitro-anisole. It is Aquat.Toxicol. 5, 315 (1984). suggested that lower value of Kow of compound in the mix- 6. Broderius S. J., v knize: Aquatic Toxicology and Risk ture lead to inhibition of toxicity of this mixture due to the Assessment (Mayes M. A., Barron M. G., ed.), 14th less accumulation of 2-nitro-anisole, which is more toxic ed. American Society for Testing and Materials, Phila- than the phenol, in an organic tissue of worms. delphia 1991. s263 Chem. Listy 101, s73−s310 (2007) 12. mezioborová česko-slovenská toxikologická konference P49 THIODIGLYCOLIC ACID – INDICATOR OF METABOLIC UNEQUILIBRIUM BRONISLAVA ŘIČAŘOVÁ, LENKA ČERNÁ Experimental Department of Occupational Diseases, First Faculty of The analysis of TDGA was carried out by the com- Medicine; General University Hospital, Charles Univer- puter-controlled Eco-Tribo Polarograph using the software sity, Na Bojišti 1, 128 00 Prague 2, Czech Republic Polar 5.1 version for Windows on hanging mercury drop email@example.com electrode (HMDE) (all Polaro-Sensors, Czech Republic). Other experiments were performed on mercury meniscus Key words: thiodiglycolic acid, TDGA, metabolism, modified or polished silver solid amalgam electrodes16−21 creatine, glutathione and on solid composite electrodes22−29 to compare30 the results with those obtained on HMDE. The process of TDGA reduction on the mercury electrode surface was Introduction studied in detail using Elimination Voltammetry with Lin- ear Scan31. For characterization of body composition and Thiodiglycolic, also thiodiacetic, mercaptodiacetic of pending parameters the multi-frequency impedance acid or dicarboxydimethyl sulfide, S(CH2COOH)2, TDGA, analyzer "In Body 3.0" was used. is one of the normal products of human metabolism and it TDGA was analyzed, after pre-separation from urine occurs at low concentrations in urine1,2. It is believed to be on a column of powdered PVC and after elution by 0.2 M formed in a number of natural metabolic pathways how- perchloric acid, by the D.C. voltammetric procedure de- ever, the most frequently studied pathway involves the scribed in1,3. The common laboratory methods were used formation of TDGA from two carbon (2C) released from for determination of other compounds determined parallel carcinogenic xenobiotics (e.g., vinylchloride monomer, with TDGA, which were useful for elucidation of meta- ethylene oxide, 1,2-dichloroethane, chloroalkyl ethers1−8). bolic pathways (in blood: uric acid, folates, vitamin B12, These 2C units are oxidized via cytochrome P450 to cholesterol, homocysteine (HoCySH), testosterone, corti- chloroacetaldehyde, which reacts with reduced GSH3,8. sone; in urine: creatine, creatinine, pH, total proteins con- From this point is this metabolic pathway common with tent). Their corrections for specific gravity were calculat- that of natural formation of TDGA from glycine over gly- ed14. oxylic and glycolic acids3,9. The process in presence of transaminases and vitamin B6 continues over S-carboxy- Results and discussion methyl-L-cysteine and S-carboxymethyl lactic acid to TDGA which in turn gets oxidized to thiodiglycol sulfox- The natural level of TDGA in urine of healthy volun- ide3. A significant increase is observed when the organism teers2 varies between 10 and 20 mg L−1. Its abnormally is exposed to the above mentioned 2C units, to the cy- increase was found in the morning urine of individuals, tostatics ifosfamide and thiotepa2,3, or to the S-carboxy- who suffered from certain metabolic disorders14. The methyl-L-cysteine10,11, or to antihistamine Zyrtec TDGA levels decreased substantially during the day. The (cetirizine dihydrochloride)2. time dependences of TDGA levels can be explained as the TDGA is also one of the final products of the degra- result of fluctuations of the daily rhythm of thiolic sub- dative pathway of thiodiglycol (TDG), which is produced stances14. Creatine, consumed regularly as a food supple- by alkaline hydrolysis of mustard gas (bis(2-chlor- ment, increases the TDGA level in the morning urine. ethylsulphide)) (MG)12,13. In accordance with the Chemi- Creatine supplemented to groups of randomly selected cal Weapons Convention a concept of chemical detoxifica- individuals, in any time of day, increased excretion of tion and complete biodegradation of organic products by TDGA rapidly within 4−8 hours following per oral intake a biotechnological method using bacterias utilizing TDG of creatine. Normal ranges of urinary TDGA levels were was studied by Ermakova et al. and Lee et al.12−14. TDGA usually reached within the following 1−2 hours. The sup- is one of the final products. Some bacterial strains can plemented creatine mostly decreased the amount of nor- therefore be used for bioremediation of MG contaminated mally excreted creatinine and increased the pH value of soils. A considerations of metabolic formation and fate of urine in the time of maximal TDGA excretion. The TDGA, structurally an analogue of MG, the microbiologi- amount of excreted TDGA is specific for different indi- cal studies on MG detoxification13 seem to offer interest- viduals. It can be supposed that supplemented creatine ing aspects. Therefore, TDGA was found in urine of Ira- reduces its endogenous production in humans. In accor- nian victims of an alleged attack with MG15. dance with previous findings2,14, vitamin B12, p.o., in- creased the TDGA level independently of creatine supple- s264 Chem. Listy 101, s73−s310 (2007) 12. mezioborová česko-slovenská toxikologická konference mentation, and did not affect the pH-value of urine. The 9. Navratil T., Petr M., Senholdova Z., Pristoupilova K., stimulation of TDGA excretion after vitamin B12 applica- Pristoupil T. I., Heyrovsky M., Pelclova D., tion is caused by disturbance of redox equilibria induced Kohlikova E.: Physiol. Res. 56, 113 (2007). by an increased supply of thiolic substances. Supplementa- 10. Hofmann U., Eichelbaum M., Seefried S., Meese C. tion of creatine increases the input of 1C and 2C units into O.: Drug Metab. Dispos. 19, 222 (1991). metabolic pathways, participated by TDGA. An increased 11. Steventon G. B.: Drug Metab. Dispos. 27, 1092 level of TDGA in urine indicates a disturbance of redox (1999). equilibria in human body. Creatine supplementation af- 12. Lee T. S., Chan S. H., Weigand W. A., Bentley W. E.: fects metabolism of thiocompounds and excretion of some Biotechnol. Prog. 16, 363 (2000). compounds into blood and urine, the metabolism of which 13. Ermakova I. T., Starovoitov I. I., Tikhonova E. B., is connected with the synthesis of creatine or with its us- Slepen'kin A. V., Kashparov K. I., Boronin A. M.: age in the body. Therefore, the levels of folates, vitamin Proc. Biochem. 38, 31 (2002). B12, homocysteine in blood, the levels of creatine, 14. Pristoupilova K., Pristoupil T. I., Navratil T., creatinine, TDGA, and pH in urine were followed and Heyrovsky M., Senholdova Z., Pelclova D.: Anal. evaluated. The results were accomplished by data of bio- Lett. 38, 613 (2005). impedance measurements. According to the changes in 15. Wils E. R. J., Hulst A. G., Vanlaar J.: J. Anal. Toxi- creatine, folates and vitamin B12 levels before and after col. 12, 15 (1988). creatine administration in course of one month, it was pos- 16. Yosypchuk B., Novotny L.: Crit. Rev. Anal. Chem. sible to divide the volunteers under study into 4 groups. 32, 141 (2002). Each of them is characterized by typical changes in levels 17. Fischer J., Barek J., Yosypchuk B., Navratil T.: Elec- of the above mentioned substances. The results confirmed troanalysis 18, 127 (2006). that creatine given as food supplement is used in human 18. Fadrna R., Yosypchuk B., Fojta M., Navratil T., body not only for creatine phosphate formation, but that it Novotny L.: Anal. Lett. 37, 399 (2004). affects the whole metabolic transformation and body con- 19. Barek J., Dodova E., Navaratil T., Yosypchuk B., stitution (mass, fat, and proteins). Novotny L., Zima J.: Electroanalysis 15, 1778 (2003). 20. Barek J., Fischer J., Navratil T., Peckova K., Yosyp- This research has been financially supported by the chuk B.: Sensors-Basel 6, 445 (2006). MSMT CR (Research Project No. 0021620807). The au- 21. Barek J., Fischer J., Navratil T., Peckova K., Yosyp- thors express their great appreciation for critical reading, chuk B., Zima J.: Electroanalysis, Accepted (2007). comments, and for language correction to Michelle Rich- 22. Navratil T., Kopanica M.: Crit. Rev. Anal. Chem. 32, ards. 153 (2002). 23. Navratil T., Kopanica M.: Chem. Listy 96, 111 REFERENCES (2002). 24. Navratil T., Kopanica M., Krista J.: Chem. Anal. 1. Dlaskova Z., Navratil T., Heyrovsky M., Pelclova D., (Warsaw) 48, 265 (2003). Novotny L.: Anal. Bioanal. Chem. 375, 164 (2003). 25. Sebkova S., Navratil T., Kopanica M.: Anal. Lett. 38, 2. Navratil T., Senholdova-Dlaskova Z., Heyrovsky M., 1747 (2005). Pristoupilova K., Pristoupil T. I., Pelclova D.: Anal. 26. Sebkova S., Navratil T., Kopanica M.: Anal. Lett. 36, Lett. 37, 1093 (2004). 2767 (2003). 3. Senholdova-Dlaskova Z.: Ph.D. Thesis. University 27. Sebkova S., Navratil T., Kopanica M.: Anal. Lett. 37, Pardubice, Pardubice 2002. 603 (2004). 4. Samcova E.: Chem. Listy 88, 723 (1994). 28. Navratil T., Senholdova Z., Shanmugam K., Barek J.: 5. Samcova E., Kvasnicova V., Urban J., Jelinek I., Electroanalysis 18, 201 (2006). Coufal P.: J. Chromatogr., A 847, 135 (1999). 29. Navratil T., Sebkova S., Kopanica M.: Anal. Bioanal. 6. Samcova E., Roth Z.: Pracovni Lekarstvi 47, 64 Chem. 379, 294 (2004). (1995). 30. Cizkova P., Navratil T., Sestakova I., Yosypchuk B.: 7. Dlaskova Z., Dvorakova L., Navratil T., Basova P.: Electroanalysis 19, 161 (2007). Chem. Listy 95, 184 (2001). 31. Sander S., Navratil T., Novotny L.: Electroanalysis 8. Ambrosi L., Soleo L., Elia G., Attimotelli R.: Suppl. 15, 1513 (2003). Med. Lav. 80, 5 (1990). s265 Chem. Listy 101, s73−s310 (2007) 12. mezioborová česko-slovenská toxikologická konference P50 STUDY OF PEROXIDASE-MEDIATED OXIDATION OF CARCINOGENIC AZO DYE SUDAN I MARCELA SEMANSKÁa, VÁCLAV MARTÍNEKa, creted in urine1, and of its oxidation by rat and human JIŘÍ HUDEČEKa, PETR HODEKa, MARTIN DRA- hepatic microsomal CYP in vitro3,4. Besides the C- ČÍNSKÝa,b, EVA FREIc, MARIE STIBOROVÁa hydroxylated metabolites, which are considered detoxica- tion products, the benzenediazonium ion (BDI) formed by a Department of Biochemistry, Faculty of Science, Charles enzymatic splitting of the azo group of Sudan I was found University in Prague, Albertov 2030, 128 40 Prague 2, to react with DNA in vitro3−6 (fig. 1). The major DNA b Institute of Organic Chemistry and Biochemistry, Acad- adduct formed in this reaction has been characterized and emy of Sciences of the Czech Republic, 166 10 Prague 6, identified as the 8-(phenylazo)guanine adduct6. This ad- Czech Republic, c Division of Molecular Toxicology, Ger- duct was also found in liver DNA of rats exposed to Su- man Cancer Research Center, 69120 Heidelberg, Ger- dan I (ref.7). Sudan I and its C-hydroxylated metabolites many are also oxidized by peroxidases, as a consequence DNA, email@example.com RNA and protein adducts are formed5,8−12 (fig. 1). While CYPs were found to be responsible for the activation of Key words: chemical carcinogenesis, Sudan I, peroxidase, Sudan I in human or animal liver, their limited role in the chromatography, mass-spectroscopy in vivo metabolic activation of Sudan I in the urinary blad- der should be taken into account. This organ has little or no detectable CYP; peroxidases are, however, present at Introduction relatively high levels in this tissue. It the case of peroxi- dase, the metabolites formed by this enzyme have, how- Sudan I [1-(phenylazo)-2-naphthalene, C.I. Solvent ever, not been identified as yet. Therefore, the present Yellow 14] was used as a food colorant in several countri- work was undertaken to isolate two major Sudan I peroxi- es1, but it has been recommended as unsafe, because it dase-mediated metabolites and characterize them partially causes tumors in the liver or urinary bladder in rats, mice, by mass spectroscopy. and rabbits, and is considered a possible carcinogen and mutagen for man1,2. Nevertheless, it is widely used to color Materials and methods materials such as hydrocarbon solvents, oils, fats, waxes, plastics, printing inks, and shoe and floor polishes1,2. Incubation mixtures contained the following in a final While the metabolism of Sudan I is not understood in hu- volume of 70 ml: 10 mM sodium phosphate buffer (pH 8.4), mans, its metabolism has been characterized in rabbits1, 0.5 µmol horseradish peroxidase (HRP), 100 µmol Sudan I where it is metabolized primarily in the liver by oxidative dissolved in methanol and 200 µmol H2O2. After incuba- or reductive reactions. Oxidation of Sudan I catalyzed by tion (37 °C, 20 min) the mixtures were extracted with ethyl cytochrome P450 (CYP) enzymes and peroxidases, was acetate (2 × 15 ml). The extracts were evaporated, dis- investigated and helped us to resolve its carcinogenic solved in a methanol, and separated on HPLC or TLC. mechanism (for a review see3). C-Hydroxylated metabo- Silica-gel TLC plates were developed in hexane-diethyl lites 4'-OH-Sudan I and 6-OH-Sudan I were found to be ether (1:3, v/v). Spots of Sudan I metabolites M1 and M2 the major products of Sudan I oxidation in vivo and ex- with Rf of 2.14 and 1.93, respectively, were extracted with methanol. Alternatively, the products were separated by HPLC on a Tessek Separon Hema S 1000 (8.0 × 250 mm) H O DNA, RNA Peroxidase 4' N N CYP1A1 C-18 column. Gradient elution (75−100 % methanol) with a flow rate of 0.3−1.5 ml min−1 was used. Sudan I metabo- (CYP3A) HO and protein adducts + HO CYP1A1 (CYP3A) Sudan I N N + lites were detected at 215, 254, 333 and 480 nm. Two Peroxidase CYP1A1 6 (CYP3A) Benzendiazonium Naphthalene-1,2-diol product peaks with r.t. of 32 and 46 min (peaks 1 and 2 in ion N H O HO N H O fig. 1) were collected. Sudan I metabolites were analyzed by mass spectroscopy. Spectra were measured using Es- N N O 6-OH-Sudan I OH 4'-OH-Sudan I + DNA O quire 3000 Bruker Daltonics (APCI, ESI – positive ioniza- tion). CYP CYP CYP 1,2-Naphthoquinone O Results and discussion HO N H O HN HO N H O N N HO N N H 2N N N N unknown + DNA adducts OH 8-Phenylazoguanine The absorption spectrum of the reaction mixture con- 4',6-diOH-Sudan I 3',4'-diOH-Sudan I in DNA taining Sudan I and peroxidase significantly changes dur- ing the Sudan I oxidation (fig. 2). During the reaction, the Fig. 1. Scheme of Sudan I metabolism absorption maximum at 480 nm (due to Sudan I) decreases s266 Chem. Listy 101, s73−s310 (2007) 12. mezioborová česko-slovenská toxikologická konference their structures have not been elucidated yet. In the present paper we used two separation procedures (TLC and HPLC) to obtain individual Sudan I metabolites in purity sufficient for their partial characterization. HPLC utilizing a Tessek Separon Hema S 1000 C-18 column was origi- nally developed here and used for purification of the two major Sudan I oxidation products (M1 and M2, fig. 3). These products were characterized by their mass spectra (fig. 4). In all cases the products decomposed during mass spectroscopy. The fragmentation peak at m/z 232 in the Fig. 2. Oxidation of Sudan I by the peroxidase/H2O2 system. metabolite M1 indicate the presence of the Sudan I mole- The samples (1 ml) contained 50 mM Na phospahte buffer pH cule without a hydroxyl group. The peak at m/z 405 sug- 8.4, 0.2 mg horseradish peroxidase, 0.15 mM Sudan I, and 0.5 mM gests the presence of the ion composed from hydroxylated H2O2. The spectra were recorded sequentially at 0 min (−−−), 2.5 Sudan I and another molecule of Sudan I, but without the min (− − −) and 10 min (. . . .) benzene ring and nitrogen atoms (fig. 4). The fragment peak at m/z 495 in the metabolite M2 indicates the ion composed of two Sudan I molecules (fig. 4). Moreover, whiles the absorbance at about 340 nm increases slightly fragmentation peaks at m/z 159, 172, 247, 389 (391) and (fig. 2). Sudan I contains a free hydroxyl group in its mole- 417 (fig. 4) seem to correspond to decomposition of the cule. Many phenolic compounds can serve as substrates Sudan I dimer molecule. Nevertheless, the real structure of for peroxidases, being oxidized to phenoxyl radicals which the both metabolites will be evaluated in detail using NMR will undergo secondary reactions in dependence on their spectroscopy. This work is under way in our laboratory. individual free radicals chemistries. This mechanism was also found by us previously for Sudan I as a substrate; Conclusion peroxidase oxidizes this carcinogen, giving rise to an oxy- gen-centered radical (naphthoxyl radical)11,12. The prod- The results of the present and previous studies9,11,12 ucts formed during peroxidase-mediated Sudan I oxidation suggest that one-electron oxidation products (radicals) are includes BDI and C-hydroxy derivatives [6-OH-Sudan I the primary intermediates in the peroxidase-mediated oxi- and 4',6-di(OH)-Sudan I]5. But major metabolites are un- dation of Sudan I. The fate of the primary free radical de- stable (sensitive to light and elevated temperature)5 and pends on the environment in which it exists. We found that Sudan I + HRP metabolites #17 200.0ul [modified by vasek] 480 nm Sudan I + HRP metabolites #21 100.0ul 480 nm 2000 Sudan I + HRP metabolites #50 TESSEK SEPARON SILIKAGEL PHENYL 480 nm a b c 2000 2000 mAU WVL:480 nm mAU WVL:480 nm mAU WVL:480 nm 63.392 1.500 1.500 800 800 800 600 600 600 1.000 1.00 1.000 1.00 400 400 400 200 200 200 Flow: 0.300 ml/min Flow: 0.300 ml/min M1 000 000 000 100.0 100.0 800 M2 800 800 600 37.492 600 M1 600 M1 32.467 400 400 400 38.277 42.323 200 200 200 %B: 0.0 % 0.0 %B: 0.0 % 0.0 63.979 min 26.388 min min 0 0 0 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 83.0 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 83.0 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 83.0 Fig. 3. HPLC of Sudan I metabolites formed by peroxidase (a) and their HPLC re-chromatography (b,c) a b Fig. 4. Mass spectra (APCI) of Sudan I metabolite M1 (a) and M2 (b) s267 Chem. Listy 101, s73−s310 (2007) 12. mezioborová česko-slovenská toxikologická konference the Sudan I reactive free radicals: (i) form additional prod- REFERENCES ucts; (ii) react with other compounds of potential physio- logical interest (reaction with NADH, ascorbate)8,9,11,12, 1. IARC Sudan I. IARC Monographs, Vol. 8, pp. 225− (iii) react with SH groups of glutathione (reducing Sudan I 231. Lyon: IARC, 1975. radicals with the formation of a thiyl radical12 and (iv) 2. Moller P., Wallin H.: Mutat. Res. 462, 13 (2000). react with macromolecules (DNA, RNA proteins) to form 3. Stiborová M., Martínek V., Rýdlová H., Hodek P., potentially toxic adducts in vitro and in vivo5,8−12. Structure Frei E.: Cancer Res. 62, 5678 (2002). elucidation of the Sudan I-(deoxy)guanosine-adducts8,11 4. Stiborová M., Asfaw B., Anzenbacher P., Lešetický will be the objective of a future study. L., Hodek P.: Cancer Lett. 40, 319 (1988). 5. Stiborová M., Asfaw B., Anzenbacher P.: FEBS Lett. Supported by grants 303/05/2195, MSM 0021620808 232, 387 (1988). and 1M4635608802. 6. Stiborová M., Asfaw B., Frei E., Schmeiser H. H., Wiessler M.: Chem. Res. Toxicol. 8, 489 (1995). Abbreviations 7. Stiborová M., Martínek V., Schmeiser H H., Frei E.: Neuro Endocrinol. Lett. 27, 35 (2006). APCI atmospheric pressure chemical ionization 8. Stiborová M., Frei E., Schmeiser H. H., Wiessler M., BDI benzendiazonium ion Hradec J.: Carcinogenesis 11, 1843 (1990). CYP cytochrome P450 9. Stiborová M., Frei E., Anzenbacher P.: Biochem. Phy- ESI electrospray-ionization siol. Pflanzen 187, 227 (1991). HRP horseradish peroxidase 10. Stiborová M., Frei E., Schmeiser H. H., Wiessler M., TLC thin layer chromatography Hradec J.: Cancer Lett. 68, 43 (1993). 11. Stiborová M., Asfaw B., Frei E.: Gen. Physiol. Bi- ophys. 14, 39 (1995). 12. Stiborová M., Asfaw B., Frei E., Schmeiser H. H.: Coll. Czech. Chem. Commun. 61, 962 (1996). s268 Chem. Listy 101, s73−s310 (2007) 12. mezioborová česko-slovenská toxikologická konference P51 CANCER MORBIDITY AND MORTALITY IN WORKERS EXPOSED TO VINYL CHLORIDE MONOMER ALENA SLÁMOVÁa, ZDENĚK ŠMERHOVSKÝb, plant. At the time when this production was launched, the EVA KUDLOVÁa effects of VCM on human organism have already been known, including the chronic and acute effects and car- a Charles University, 1st Faculty of Medicine, Institute of cinogenic hazards. All available information and hygienic Hygiene & Epidemiology, Studničkova 7, 128 00 Prague instructions for the staff of the district hygienic station of 2, b National Institute of Public, Šrobárova 48, 100 42 the Central Bohemian locality that were in charge of the Praha 10 plant's hygienic supervision ensure efficient environ- firstname.lastname@example.com mental health protection of the workers. C h a r a c t e r i s t i c s of t h e p r o d u c t i o n a n d Key words: vinyl chloride-monomer (VCM), demon- the workplace conditions strated chemical carcinogen, standardised mortality ratio VCM is produced in hermetically sealed columns in Introduction the open. The initial substance is ethylene and chlorine. The maximum acceptable concentration (MAC) mean is The production of the most popular plastic material − 10 mg m−3, MAC limit is 30 mg m−3 – until June 2001 and polyvinyl chloride (PVC) was started in 1933 and during 20 mg m−3 afterwards. Values measured at workplaces the past 60 years has been applied in almost every indus- were usually under the MAC value limits; only exception- trial activity. ally they were higher but never exceeded 100 mg m−3. According to IARC Lyon Agency monograph of 1987 VCM is classified I. group as a demonstrated carcinogen3 Malignant tumours with target effects on the liver2. Exposed workers showed significantly increased incidence of liver tumours, mostly Vinyl chloride pilot plant production was started in angiosarcoma (ASL), insignificant mortality rise for tu- 1974 full run production in 1977. Since 1974 until the end mours in general, in particular higher incidence lung1, of 2003, a total of 908 employees worked in the PVC pro- brain cancers, and lymphosarcoma4. duction plant with an average exposure of 9.4 years (SD 9.09). There were a total of 208 women with an average Materials and methods exposure of 6.7 (0.1–28.8) years and 700 men with an average exposure of 9.9 (0.1−29) years. Comparison of the Data about workers employed in the vinylchloride cohort data with the National Cancer Register revealed 44 manufacturing plant from 1974 until the end of 2003 were workers (15 women and 29 men) who were diagnosed obtained from the factory card file. The health status study with tumour disease by the end of 2003. Twenty seven of of this manufacturing plant was designed as a retrospective them died (6 women and 21 men). Angiosarcoma was not cohort study exploiting data on the Czech population diagnosed, hepatoma was diagnosed twice, carcinoma of health status provided by Institute of Health Information pancreas three times, gall bladder twice, and lung and and Statistics of the Czech Republic (IHIS) and National bronchi nine times (data about smoking were not avail- Cancer Register (NCR) also administered by IHIS. Mortal- able). Analysis of malignant tumours morbidity and mor- ity and morbidity of exposed workers was compared with tality (MT) has shown that the observed MT mortality (27) an external control group, which constituted total Czech is statistically significantly different from the expected one population. Data on the incidence of monitored phenom- (10.7) in men – 21 observed deaths vs. 9.37 expected ena specific for sex, five-year age groups, and calendar (SMR 1.43; 95 % CI 1.43−3.36) as well as in women – 6 years were used in calculations. Indirect standardization observed deaths vs. 1.35 expected (SMR 4.44; 95 % CI was used to control potentially confounding effects of sex 2.18−8.26). Similar results were found also for total mor- and age; changes in the morbidity a mortality trends in tality and MT incidence (Table I). particular calendar periods were also taken into considera- tion. Discussion Results Analysis results indicate statistically significantly increased deviation of the health status of observed/ V C M p r o d u c t i o n in C z e c h R e p u b l i c followed up cohort of workers exposed to vinylchloride in comparison with the Czech population health status re- VCM, the polymerization of which produces polyvi- garding total mortality as well as tumour mortality and nylchloride is in the Czech Republic produced by a single morbidity. We are very careful in their interpretation as s269 Chem. Listy 101, s73−s310 (2007) 12. mezioborová česko-slovenská toxikologická konference Table I Standardized cancer mortality ratios according to calendar years CANCER OCCURENCE EXPECTED OBSERVED FEMALE MALE Year Female Male F+M Female Male SMR 95% Cl Sig. SMR 95% Cl 1980 0.022 0.147 0.169 1 0 45.26 4.10 211.02 0.012 0.00 0.809 1981 0.020 0.152 0.172 0 0 0.00 0.254 0.00 0.823 1982 0.028 0.166 0.194 0 0 0.00 0.399 0.00 0.857 1983 0.030 0.181 0.211 0 0 0.00 0.411 0.00 0.890 1984 0.026 0.223 0.249 0 0 0.00 0.391 0.00 0.977 1985 0.031 0.243 0.274 1 2 32.33 2.93 150.73 0.017 8.23 1.64 26.37 0.018 1986 0.039 0.276 0.316 0 0 0.00 0.456 0.00 0.927 1987 0.048 0.320 0.367 0 1 0.00 0.496 3.13 0.28 12.17 0.227 1988 0.054 0.331 0.385 0 3 0.00 0.523 9.07 2.51 24.19 0.004 1989 0.059 0.366 0.425 0 2 0.00 0.543 5.47 1.09 17.53 0.042 1990 0.069 0.415 0.484 0 2 0.00 0.579 4.82 0.96 13.31 0.054 1991 0.078 0.441 0.519 1 1 12.90 1.17 60.15 0.042 2.27 0.21 10.57 0.377 1992 0.074 0.442 0.516 0 1 0.00 0.597 2.26 0.21 10.54 0.338 1993 0.087 0.442 0.529 0 1 0.00 0.642 2.26 0.21 10.55 0.338 1994 0.073 0.481 0.554 1 0 13.74 1.24 64.04 0.039 0.00 0.656 1995 0.079 0.474 0.553 0 0 0.00 0.615 0.00 0.663 1996 0.064 0.495 0.559 0 0 0.00 0.562 0.00 0.672 1997 0.069 0.528 0.597 0 1 0.00 0.579 1.90 0.17 8.84 0.418 1998 0.063 0.530 0.593 0 1 0.00 0.558 1.89 0.17 8.80 0.420 1999 0.061 0.427 0.487 1 0 16.50 1.50 76.94 0.032 0.00 0.716 2000 0.060 0.431 0.491 0 1 0.00 0.547 2.32 0.21 10.81 0.328 2001 0.058 0.441 0.499 0 3 0.00 0.537 6.80 1.88 18.13 0.009 2002 0.071 0.482 0.553 0 2 0.00 0.585 4.15 0.83 13.30 0.073 2003 0.029 0.426 0.455 1 0 35.04 0.015 0.00 0.717 Total 1.353 9.372 10.725 6 21 4.44 2.18 8.26 0.003 2.24 1.43 3.36 0.001 evaluating the exposure of workers is very complicated REFERENCES because the individual length and intensity of exposures are very different. 1. Maroni M., Fanetti C. A: Int. Arch. Occup. Environ. All employees were included into the study even Health 79, 57 (2006). those not expected to have been exposed to carcinogens or 2. Mastrangelo G., Fedeli U., Prilo G., Buja A.: Cancer those who worked at the worksite for one month only. We Causes Control. 16, 189 (2005). did not exclude these employees from the sample due to 3. Vinyl chloride. IARC monographs on the evaluation the total sample size. We have not calculated standardized of carcinogenic risks to humans. 7, 1 (1987). indexes for particular types of tumour diseases due to their 4. Ward E., Boffetta P., Andersen A.: Epidemiol. 12, 710 small numbers. (2001). s270 Chem. Listy 101, s73−s310 (2007) 12. mezioborová česko-slovenská toxikologická konference P52 FREQUENCY OF TPMT FUNCTIONAL POLYMORPHISMS IN THE CZECH POPULATION ONDŘEJ SLANAŘ, HELENA BUZKOVÁ, Methods KRISTÍNA PECHANDOVÁ, JOSEF MAREK, FRANTIŠEK PERLÍK Two hundred and sixty-seven young healthy volun- teers (161 men and 106 women, aged 18−56 years) were Charles University in Prague, 1st Faculty of Medicine, De- included in the study after obtaining their written informed partment of Pharmacology, Na Bojišti 1, 120 00 Praha 2 consent. All subjects participating in the study were unre- firstname.lastname@example.com lated subjects of Czech nationality. Peripheral blood samples were collected from all Key words: pharmacogenetics, single nucleotide polymor- volunteers in 7 ml collecting tubes with ethylenediamine- phism, thiopurine S-methyltransferase tetraacetic acid (EDTA). Genomic DNA was subsequently isolated by a standard phenol-chlorophorm method and stored at 4 °C until analysis. Introduction PCR amplification was run in a MyCycler (Bio-Rad, USA) using primers as described previously and specified Azathioprine and its metabolite 6-mercaptopurine are in Table I. Subsequent RFLP analysis produced fragments thiopurines with the latter acting as a purine antimetabolite specified in Table I, allele specific PCR was run for which can inhibit purine biosynthesis by both de novo and TPMT*2. The fragments were separated in 3 % agarose salvage pathways. Metabolically, 6-mercaptopurine is gel and visualized by staining with ethidium bromide. The converted by hypoxanthine phosphoribosyltransferase to primers were ordered at Sigma-Aldrich (St. Louis, USA), activated 6-thioguanine nucleosides. These latter interme- all other components of PCR reaction mix and Top Vision diates are ultimately incorporated into DNA as false bases. agarose were purchased from Fermentas (Lithuania). Inactivation of 6-mercaptopurine and 6-mercaptopurine The study was approved by the Ethics Committee of nucleosides is mediated by methylation with thiopurine S- the General Teaching Hospital in Prague. methyltransferase (TPMT) to inactive metabolites methyl- Expected genotype frequencies were calculated using mercaptopurine or methylmercaptopurine nucleosides, Hardy–Weinberg equilibrium from the observed allelic respectively. TPMT is well characterized cytosolic en- frequencies. Prevalence was compared by the chi-square zyme, with several functionally important genetic poly- test. Microsoft Excel 8.0 (Microsoft, USA) and Statgraph- morphisms leading to decreased enzyme activity. ics Plus 3.1 (StatPoint, Inc., USA) were used for data han- There is substantial amount of evidence, which shows dling. that individual activity of TPMT is one of the major fac- tors for wide variation in the metabolism, the drug effi- Results cacy, and mainly severe toxicity of thiopurine drugs1−5. The patients, who have low or no detectable enzyme activ- Table II shows observed genotypes and respective ity, suffer more frequently from myelotoxicity when allelic frequencies in our study. The most frequent allele treated with standard doses of azathioprine. On the other was TPMT*3A, while no subject carrying TPMT*3B al- hand, subjects with high activity of the enzyme could have lele was found. Totally 26 (9.7 %) subjects were found to reduced clinical effect of the treatment. Genetic basis for be heterozygous carriers of one of the variant alleles and 1 such a variation in TPMT activity is known only for defi- (0.4 %) volunteer was homozygous poor metabolizer. The cient states while no corresponding factors for ultra-rapid distribution of genotypes was similar to the predicted num- type of TPMT metabolism were found so far6. bers. Table I Sequences of the primers used, restriction enzymes and discrimative lenghths of fragments [bp] Polymorphism Primer name - sequence Restriction enzyme Fragment size G460A P460F 5'-ATAACAGAGTGGGGAGGCTGC P460R 5'-CTAGAACCCAGAAAA AGTATAG Acc I 293/207 A719G P719R 5'-TGTTGGGATTACAGGTGTGAGCCAC P719F 5'-CAGGCTTTAG CATAATTTTCAATTCCTC Mwo I 365/267 G238C P2W 5'-GTATGATTTTAT GCAGGTTTG P2M 5'-GTATGATTTTATGCAGGTTTC P2C 5'-TAAATAGGAACCATCGGACAC − 254 s271 Chem. Listy 101, s73−s310 (2007) 12. mezioborová česko-slovenská toxikologická konference Table II Number of subjects with specific TPMT genotypes and respective allelic frequencies Polymorphism Observed genotypes Alelic frequencies [%] w/w w/v v/v w v TPMT*2 266 1 0 99.82 0.18 TPMT*3A 244 22 1 95.52 4.48 TPMT*3B 267 0 0 100.00 0.00 TPMT*3C 264 3 0 99.44 0.56 w − wild-type allele, v − variant allele Discussion European countries. These results also provide basal infor- mation for future pharmacogenetic and pharmacoeconomic Similarly to our findings, variant allele TPMT*3A studies in the Czech population. was reported as the most prevalent deficient allele in other Caucasian populations, whereas TPMT*3C is the most This study has been supported by grants No. IGA MZ frequent variant in Asian populations7,8. ČR NR9094, and MSM0021620849. A good correlation between high concentrations of activated 6-thioguanine nucleosides and severe forms of REFERENCES bone marrow suppression in TPMT homozygous poor metabolizers treated with usual doses of azathioprine has 1. Duley J. A., Florin T. H.: Ther. Drug. Monit. 27, 647 been well established. Some studies revealed similar pat- (2005). tern of relationship between high levels of 6-thioguanine 2. Breen D. P., Marinaki A. M., Arenas M., Hayes P. C.: nucleosides and clinical effects in heterozygous intermedi- Liver Transpl. 11, 826 (2005). ate metabolizers, but other authors did not report such 3. Krynetski E.Y., Evans W.E.: Pharmacology 61, 136 findings. Although there exists such a discrepancy be- (2000). tween the data a screening for TPMT deficient patients 4. Frohman E. M., Havrdova E., Levinson B., Slanar O.: prior to beginning of azathioprine therapy seems to be cost Mult. Scler. 12, 108 (2006). effective approach9,10. Our data provide necessary basis, 5. Rosen R., Integlia M. J.: J. Pediatr. Gastroenterol. for transferring the observed individual, social and eco- Nutr. 35, 695 (2002). nomic consequences of TPMT deficiency from other Cau- 6. Yates C. R., Krynetski E. Y., Loennechen T., Fessing casians into the Czech population. M. Y., Tai H. L., Pui C. H., Relling M. V., Evans W. E.: Ann. Intern. Med. 126, 608 (1997). 7. Tavadia S., Mydlarski P., Reis M., Mittmann N., Pin- Conclusions kerton S. N.: J. Am. Acad. Dermatol. 42, 628 (2000). 8. Marra C., Esdaile J., Anis A.: J. Rheumatol. 29, 2507 Our results show similar distribution of the most fre- (2002). quent variant TPMT alleles in comparison to other Cauca- 9. Collie-Duguid E. S., Pritchard S. C., Powrie R. H.: sian populations. Therefore the consequences of azathio- Pharmacogenetics 9, 37 (1999). prine therapy arising from TPMT deficiency can be ex- 10. Kumagai K., Hiyama K., Ishioka S.: Pharmacogenet- pected similar proportions as described in some other ics 11, 275 (2001). s272 Chem. Listy 101, s73−s310 (2007) 12. mezioborová česko-slovenská toxikologická konference P53 OXIDATIVE DAMAGE AND ANTIOXIDANT DEFENCE IN THE PLASMA OF PEOPLE EXPOSED TO MINERAL FIBRES MARTA STARUCHOVÁa, KATARÍNA investigated (21 to 58 years old), 43 controls (20 men, 23 VOLKOVOVÁa, CSILLA MIŠĽANOVÁa, ZUZANA women and 27 non-smokers, 16 smokers) and 98 exposed KOVÁČIKOVÁa, LADISLAVA WSÓLOVÁa, (75 men, 23 women and 61 non-smokers, 37 smokers). A. R. COLLINSb, LADISLAV STARUCHc, The rock wool exposure lasted at least 5 years. MÁRIA DUŠINSKÁa,d All subjects contributed a single blood donation in autumn. A urine sample was used for measurement of a Research Base of Slovak Medical University, Limbová cotinine to determine smoking status. Blood was collected 12, 833 03 Bratislava, b Institute for Nutrition Research, by venipuncture from fasted subjects, using an anticoagu- University of Oslo, Norway, c Faculty of Chemical and lant as EDTA used for isolation of lymphocytes and eryth- Food Technology,Slovak University of Technology, Brati- rocytes. The samples of plasma, lymphocytes and lympho- slava, d Norwegian Institute of Air Research (NILU), cyte extracts were stored on ice until assayed or were fro- Kjeller, Norway zen at −80 °C for various biochemical measurements. email@example.com M e a s u r e me n t o f D N A d a m a g e a n d r e p a i r Keywords: Rockwool, oxidative DNA damage, DNA re- The comet assay (single cell alkaline gel electropho- pair; antioxidative enzymes resis)5 was applied to freshly isolated lymphocytes (Lymphoprep, Nycomed, Oslo, Norway) for measurement Introduction of DNA strand breaks, oxidized bases and DNA alkyla- tion6,7. The assay for OGG1 activity5 was used to measure Oxidative damage of biomolecules (DNA, proteins, the ability of a cell-free lymphocyte extract to incise sub- lipids) caused by free radicals is involved in the patho- strate DNA containing 8-oxoGua6,7. genesis of different diseases such as cancer, atherosclero- sis, inflammation, etc.1 Cells respond to toxic levels of M e a s u r e me n t o f a c t i v i t i e s o f a n t i o x i d a n t reactive oxygen species (ROS) by activating a diverse e n z y me s arrays of protective responses. This includes a complex range of enzymatic antioxidants such as catalase (CAT), Erythrocytes were washed three times with isotonic glutathione peroxidase (GPX), superoxide dismutase saline. Isolated erythrocytes were used for measurement of (SOD), glutathione-S-transferase (GST) and non- activities of antioxidant enzymes. The activity of glu- enzymatic antioxidants such as glutathione, tocopherols tathione peroxidase (GPx) was determined indirectly by and carotenoids2. Oxidative repair mechanisms, includes oxidation of NADPH to NADP+ measured by the kinetic DNA repair, protein and lipid repair, is degradation path- method according to Paglia and Valentine8. Catalase ways3. (CAT) was measured spectrophotometrically by a modi- The adverse effect that arises from exposure to asbes- fied method of Cavarocchi et al.9 and glutathione-S- tos has stimulated an extensive research into the develop- -transferase (GST) by a kinetic method according to Habig ment of substitute materials. One such substitute is rock- et al10. The activity of superoxide dismutase (SOD) was wool, made from natural basic rock material, which is used estimated by a commercial test (Randox lab, Ltd., U.K.). mainly as isulation for thermal, acoustic and fire protection The index of the combined non-enzymatic antioxidant of roofs, walls and floors. However, little is known about capacity of plasma (FRAP) was measured spectropho- the health effects of these fibres. The potentially harmful tometrically according to Benzie and Strain11 ferric to fer- effects of all types of respirable fibres are at present one of rous ion reduction at low pH causes formation of a col- the most important fields of interest in industrial hygiene4. oured ferrous–tripyridyltriazine complex. Ceruloplasmin oxidase activity in plasma was assayed Subjects and methods with the use of o-dianisidine dihydrochloride according to the method of Schosinsky et al.12 In order to study the effect of rockwool exposure on Plasma vitamin C was detected by HPLC13. Lipid oxidative DNA damage and lipid peroxidation, an epide- peroxidation was determined by the levels of malondialde- miological study was conducted in a rockwool factory in hyde (MDA) using a modified HPLC method in plasma14. Slovakia. To examine the interactions between fibre- and ciga- Results and discussion rette smoke- induced effects, the cohorts examined in- cluded non-smokers as well as individuals smoking differ- Rockwool exposure induced elevation of DNA strand ent numbers of cigarettes. Altogether 141 subjects were breaks in the lymphocytes of investigated subjects (P= s273 Chem. Listy 101, s73−s310 (2007) 12. mezioborová česko-slovenská toxikologická konference 0.05). When analysed according to sex and smoking habit, molecules especially in the group of male non-smokers; this effect was apparent only in the group of non smokers. however, the optimal levels of antioxidants could have DNA strand breaks were higher in exposed subjects com- a protective effect. pared to controls (P=0.004). The effect of occupational The work was supported by the E.C. grant, exposure to rockwool on oxidative DNA damage and re- FIBRETOX contract No. QLK4-CT-1999-0162. pair was already published by Dusinska et. al.15. DNA repair of oxidative damage in lymphocytes of exposed REFERENCES subjects was higher in group of men (P=0.02) compared to women, and controls (P=0.07). 1. Ames B. N., Shigenaga M. K., Hagen T. M.: Proc. We found higher MDA levels in the group of all ex- Natl. Acad. Sci.U.S.A 90, 7915 (1993). posed workers (P=0.025) and in exposed non-smokers 2. Choudhary S., Xiao T., Srivastava S., Zhang W., Chan (p=0.003) possibly as the consequence of significantly L. L., Vergara L. A., Van Kuijk F. J., Ansari N. H.: suppressed activity of CPL-oxidase (P=0.02, 0.016 respec- Toxicol. Appl. Pharmacol. 204, 122 (2005). tively) and CAT in these groups (P=0.04, 0.012 respec- 3. Evans M. D., Dizdaroglu M., Cooke M. S.: Mutat. tively). The activity of GST was affected by exposure to Res. 567, 1 (2004). rockwool; the GST levels were significantly lower in the 4. Man-made Viterous Fibres, IARC Monographs, vol. all exposed subjects (P=0.04), in the exposed non-smokers 81, IARC, Lyon 2002. (P=0.03), and exposed men (P=0.007). Concentration of 5. Collins A. R., Dusinska M., Horvathova E., Munro E., vitamin C in plasma and the FRAP were not affected by Savio M., Stetina R.: Mutagenesis 16, 297 (2001). the rockwool exposure. 6. Collins A. R.: Mol. Biotech. 26, 249 (2004). There was a significant negative correlation between 7. Dusinska M., Collins A., Kazimirova A., the activity of GPX and MDA in the whole group Barancokova M., Harrington V., Volkovova K., (P=0.007) and in the exposed group, and between CAT Staruchova M., Horska A., Wsolova L., Kocan A.: activity and MDA in the all subjects (P=0.009). We found Mutat. Res. 553, 91 (2004). inverse correlations between activities of several antioxi- 8. Paglia D. E., Valentine W. N.: J. Lab. Clin. Med. 70, dant enzymes (GPX, GST, and CAT) and DNA damage. 58 (1967). GST activity correlated inversely with oxidised purines 9. Cavarocchi N. C., England M. D., O'Brien J. F., Solis measured as Endonuclease sensitive sites almost in all E., Russo P., Schaff H. V., Orszulak T. A., Pluth J. R., subgroups: in all subjects (P=0.002), in exposed (0.014), Kaye M. P.: J. Surg. Res. 40, 519 (1986). men (0.038), women (0.004), and exposed men (0.041). 10. Habig W. H., Pabst M. J., Jakoby W. B.: J. Biol. We found a significant negative correlations between Chem. 249, 7130 (1974). DNA repair and GPX in all subjects as well as in control 11. Benzie I. F. F. and Strain J. J.: Anal. Biochem. 239, men (P=0.03, 0.028 respectively) and CAT in all control 70 (1996). subjects and control men (P=0.019, 0.009 respectively). 12. Schosinsky K. H., Lehmann H. P., Beeler M. F.: Clin. Interestingly we found the positive correlations between Chem. 20, 1556 (1974). DNA repair and MDA in all subjects and in all exposed 13. Cerhata D., Bauerová A., Ginter E.: Ceska Slov. (P=0.008, 0.026 respectively). Farm. 43, 166 (1994). 14. Richard M. J., Guiraud P., Meo J., Favier A.: J. Chro- Conclusion matogr. 577, 9 (1992). 15. Dusinska M., Barancokova M., Kazimirova A., Har- The presented results indicate that rockwool exposure rington V., Volkovova K., Staruchova M., Horska A., induces an increase in the oxidative damage of bio- Wsolova L., Collins A.: Mutat. Res. 553, 103 (2004). s274 Chem. Listy 101, s73−s310 (2007) 12. mezioborová česko-slovenská toxikologická konference P54 TLC PROOF OF INTOXICATION BY TAXUS BACCATA JAN STŘÍBRNÝa,b, MICHAL DOGOŠIa, ZDENĚK sis: blood, urine, samples of the brain and lung tissue, sam- ŠŇUPÁREKa, IVANA ČERNÁa, MILOŠ SOKOLa ples of the liver and kidney tissue and stomach and duode- num content (pH 8). No blood alcohol was detected. Nor a Vojenský ústav soudního lékařství ÚVN, U vojenské any volatile toxic compounds in the brain and lung tissue nemocnice 1200, 169 02 Praha 6, b Fakulta vojenského were found. Toxicological analysis using GC/MS detected zdravotnictví Univerzity obrany, Hradec Králové carboxy-tetrahydrocannabinol, caffeine and nicotine in the firstname.lastname@example.com blood. Morphological and anatomical evaluation of the mash plant material found in the stomach content detected the presence of Taxus baccata leaves (evaluation per- Key words: Taxus, Taxine, TLC, intoxication formed by RNDr. Dagmar Nová). Introduction Materials and methods The toxicity of yew (Taxus baccata) has been known Material used since antiquity. It was believed that as "a tree of death" it endangered lives of those who stayed in its shade for Mixture of taxin B and isotaxin B prepared in the a longer time. Yew was used in homicide, suicide, and as Institute of Legal Medicine, University Hospital Münster. an abortive. All parts of yew, except for the red epicarp of Chromatographic plates Kieselgel 60 F254 Merck, Fast the berries, are toxic. The toxicity of yew is attributed to Blue B reagent (FBB), Ehrlich reagent, Drägendorff re- taxin, a mixture of several pseudoalkaloids, in which the agent, ethanol 96 %, organic solvents used in extraction evidently dominant components are taxin B and isotaxin and preparation of mobile phases were p.a. purity, ammo- B. These taxin alkaloids are cardiotoxic. Their effect can nium hydroxide solution 26 % p.a., hydrochloric acid be determined as soon as 1/2 hour after ingestion. Death conc. p.a. after 2 and 1/2 hours after ingestion of two handfuls of yew leaves was reported1. E x t r a c t i o n o f c o mp a r a t i v e p l a n t ma t e r i a l Description of toxicological proofs of yew poisoning is not very frequent in literature. There is a description of 100 ml of 3.7 % hydrochloric acid was added to 50 g intoxication evidence by thin layer chromatography2,3, of of Taxus baccata leaves, the mixture was homogenized the evidence achieved by chromatogram similarities be- and macerated for 1 hour, then filtered. The filtrate was tween yew extract and extract of stomach content4 (GC/ extracted twice with 250 ml diethyl ether and then aIka- FID), of the finding of 3,5-dimethoxyphenol by gas chro- lized to pH 10 with sodium hydroxide and extracted twice matography5 (3,5-dimethoxyphenol is considered as with 250 ml diethyl ether. The acidic and basic extract was a marker of yew ingestion) and the evidence of taxin B and evaporated and the evaporates dissolved in 1.5 ml ethanol. isotaxin B determined in biological material by liquid chromatography with mass detector6,7. E x t r a c t i o n o f s e c t i o n ma t e r i a l In the following paragraphs the authors describe a case of lethal intoxication caused by the decoction of Samples of liver and kidney tissue (50 g) were ho- yew leaves, and the proof of it by thin layer chromatogra- mogenized, 24 hours soaked in acetone and ethanol mix- phy (TLC). ture (450 ml, 7:3) and then the macerate was filtered and evaporated to 50 ml. The stomach content was diluted with Case report distilled water and filtered. Urine, the macerate of the liver and kidney tissue, the filtrate of the stomach content and A 28-year-old man was found dead in the family the found brown liquid were acidified with hydrochloric house. For toxicological analysis the police supplied acid to pH 3 and extracted 2 times with 250 ml diethyl a brown liquid (pH 9) found in a bottle and a glass in the ether. The water fractions were subsequently alkalized to living room. Forensic autopsy stated the following: acute pH 10 with the solution of sodium hydroxid and extracted catarrhal esophagitis, acute gastritis, a mash of plant mate- with 250 ml diethyl ether and 250 ml chloroform. Acidic rial in the stomach and duodenum (in dry form 3.6 g), and basic extracts were vaporized and consequently dis- epicranial, pleural and epicardial pete-chiae, brain swell- solved in 1.5 ml ethanol. Further, urine was hydrolyzed ing, haemorrhagical swelling of the lungs, acute venostasis and after neutralization extracted with 250 ml diethyl of the parenchymatous organs, and liquid status of the ether. blood. The following was supplied for toxicological analy- s275 Chem. Listy 101, s73−s310 (2007) 12. mezioborová česko-slovenská toxikologická konference M o b i l e p h a se The Drägendorff reagent reacts in colour with the taxin B / isotaxin B standard. In chromatographic condi- A ethyl acetate : ethanol : ammonium hydroxide solu- tions mentioned above both the compounds do not sepa- tion 26 % = -36:2:2 rate and only one spot on the chromatogram appears B chloroform : ethanol : ammonium hydroxide solu- (RfA=0.6; RfB=0.4). A presence of taxin B / isotaxin B tion 26 % = 38:2:1 / paper was proved in the basic extract of stomach content. In the liver and kidney tissue extracts, as well as in the urine and Abbreviations urine hydrolysate a presence of taxin B / isotaxin B was not detected. RfA retention factor in mobile phase A RfB retention factor in mobile phase B Conclusion Results On the basis of the described results, the authors as- sume that the intoxication with Taxus baccata can be Fast BlueB detection proved analytically by the TLC method. With detection of Fast Blue B and Ehrlich reagent, compounds of corre- Some substances from acidic extract of yew show a sponding character with substances contained in Taxus very prominent colour reaction with the FBB reagent. In baccata were identified. The presence of these substances acidic extracts of stomach content, urine and the found was proved in the stomach and duodenum content and brown liquid FBB reagent proved a presence of substances urine, whereas it was not detected in the liver and kidney of corresponding character with substances contained in tissue and urine hydrolysate. The Drägendorff reagent the extract of Taxus baccata. A bright orange-red spot in reacts with a number of substances present in the yew the position RfA=0.8; RfB=0.4 was particularly persua- leaves extract, particularly with substances contained in sive. A presence of substances contained in Taxus baccata the extract from basic water environment. Some spots on extract was not determined in the liver and kidney tissue the chromatogram of the yew basic extract correspond nor in the urine hydrolysate. FBB reagent showed no col- with the spots on the chromatogram of the stomach and our reaction with taxin B / isotaxin B standard. duodenum content. The chromatogram is not clear enough because of multiple spots. The comparision of biological Ehrlich reagent detection material extracts with the taxin B / isotaxin B standard proved that one of the spots reacting positively with the With the Ehrlich reagent some substances from acidic Drägendorff reagent on the chromatogram of the basic and basic extracts of yew show a prominent colour reac- extract of stomach and duodenum content is taxin B / iso- tion after heating the chromatographic plate. In acidic and taxin B. Under the stated chromatographic conditions there basic extracts of the stomach content and urine Ehrlich was no separation of taxin B from isotaxin B. reagent proved a presence of substances of corresponding character with substances contained in the extract of Taxus The authors wish to thank Dr. J. Beike from Univer- baccata. Particularly persuasive was a pink spot on the sity Hospital Münster for supplying the taxin B / isotaxin B chromatogram of the acidic extract in position RfA=0.8; standard. RfB=0.4. After heating the plate, intensified spots of blue- violet colour showed on the chromatogram of basic extract of yew, and two of them also appeared in the basic extract REFERENCES of stomach content (RfAl/A2=0.7/0.6; RfBl/B2=0.5/0.4). A pre-sence of substances contained in Taxis baccata ex- 1. Kubis M., Kosatík A., Srp L., Švejda J., Mařík M.: tract was not determined in the liver and kidney tissue nor Prakt. Lék. 60, 720 (1980). in the urine hydrolysate. Ehrlich reagent showed no colour 2. Kosatík A., Smysl B.: Scripta medica 51, 118 (1978). reaction with taxin B / isotaxin B standard. 3. Vaněrková H., Lysenková A.: Folia Fac. Med. Univ. Comenianae Bratisl, Suppl. 1990, 157. Drägendorff reagent detection 4. Sinn L. E., Porterfield B. S., Porterfield J. F.: J. Foren- sic Sci. 36, 599 (1991). Substances contained in basic and acidic extracts of 5. Musshoff F., Jacob B., Fowinkel C., Daldrup T: Int. J. yew react with the Drägendorff reagent. Some spots on the Legal Med. 106, 45 (1993). chromatogram of basic extract of yew correspond with 6. Beike J., Karger B., Meiners T., Brinkmann B., spots on the chromatogram of basic extract of stomach Köhler H.: Int. J. Legal Med. 117, 335 (2003). content.. Because of multiple spots the chromatogram is 7. Frommherz L., Kintz P., Kijewski H., Köhler H., less clear. A presence of substances contained in Taxus Lehr M., Brinkmann B., Beike J.: Int. J. Legal Med. baccata extract was not determined in the liver and kidney 120, 346 (2006). tissue nor in the urine. s276 Chem. Listy 101, s73−s310 (2007) 12. mezioborová česko-slovenská toxikologická konference P55 REDUCTIVE ACTIVATION OF ENVIRONMENTAL POLLUTANTS 3-NITROBENZANTHRONE AND 2-NITROBENZANTHRONE MARTINA SVOBODOVÁa, JANA ŠÍSTKOVÁa, mented by the detection of specific DNA adducts formed HELENA DRAČÍNSKÁa, JIŘÍ HUDEČEKa, PETR in vitro as well as in vivo in rodents1,4−7. Most of the meta- HODEKa, HEINZ H. SCHMEISERc, VOLKER bolic activation of 3-NBA in vitro is attributable to human M. ARLTb, EVA FREIc, MARIE STIBOROVÁa and rat cytosolic NAD(P)H:quinone oxidoreductase (NQO1), while human N,O-acetyltransferase (NAT), a Department of Biochemistry, Faculty of Science, Charles NAT2, followed by NAT1, sulfotransferase (SULT), University, Albertov 2030, 128 40 Prague 2, Czech Repub- SULT1A1 and, to a lesser extent, SULT1A2 are the major lic, b Section of Molecular Carcinogenesis, Institute of phase II enzymes activating 3-NBA (ref.7). Microsomal Cancer Research, Surrey SM2 5NG, United Kingdom, NADPH:cytochrome P450 (CYP) reductase is also effec- c Department of Molecular Toxicology, German Cancer tive in the activation of 3-NBA (ref.6), but in a model or- Research Center, 69 120 Heidelberg, Germany ganism, mice, 3-NBA is predominately activated by cyto- email@example.com, firstname.lastname@example.com solic nitroreductases such as NQO1 rather than micro- somal NADPH:CYP reductase7 (fig. 1). Key words: 3-nitrobenzanthrone, 2-nitrobenzanthrone, While 3-ABA was suggested to be the main reductive reduction, NAD(P)H:quinone oxidoreductase, HPLC metabolite of 3-NBA (ref.7), the reactions of 3-NBA to 3- ABA by enzymatic systems in vitro and in vivo has not been investigated as yet. Reductive metabolism of 3-NBA Introduction is, therefore, investigated in this work. In addition, reduc- tion of 3-NBA is compared with that of its isomer 2-NBA. 3-Nitrobenzanthrone (3-nitro-7H-benz[de]anthracen- -7-one, 3-NBA, fig. 1), occurs in diesel exhaust and in Materials and methods airborne particulate matter1. 3-NBA might originate both from incomplete combustion of fossil fuels and from reac- 3-NBA, 2-NBA, 3-ABA and N-OH-ABA were syn- tion of the parent aromatic hydrocarbon with nitrogen ox- thesized as described1,2,8. Incubations with human recom- ides in the atmosphere. 3-NBA can spontaneously isomer- binant NQO1 or rabbit NADPH:CYP reductase, in a final ize to 2-nitrobenzanthrone (2-nitro-7H-benz[de]anthracen- volume of 500 µl, consisted of 50 mM Tris-HCl buffer -7-one, 2-NBA), which can become more than 70-fold (pH 7.4), containing 0.4 % Tween 20, 1 mM NADPH, higher in concentration in ambient air1,2. 3-Amino- from 5 to 50 µM 3-NBA or 2-NBA (in DMSO) and from benzanthrone (3-ABA, fig. 1), suggested to be the main 10 to 100 µg ml−1 of NQO1 or NADPH:CYP reductase. In reductive metabolite of 3-NBA, has been found in urine incubations testing the time-dependent formation of 3- samples of salt mine workers occupationally exposed to NBA-DNA adducts mediated by human recombinant diesel emissions3, demonstrating that human exposure to NQO1 (20 µg ml−1), incubation times varied between 15 3-NBA in diesel emissions can be significant and is detect- and 60 minutes and 20 mM 3-NBA were used. The incu- able. 3-NBA is carcinogenic in rats, causing lung tumours bation mixtures were extracted with ethyl acetate (2 × after intratracheal instillation4. It is also an exceptionally 1 ml) and 5 µl of 1 mM phenacetine was added as an inter- potent mutagen1. Its genotoxicity has been further docu- nal standard. The extracts were evaporated to dryness; H H H N O R N N + DNA dG-N2-ABA dG-C8-N-ABA dA-N6-ABA O O O human NATs human SULTs plant HRP human MPO rat and human (NAT1, (SULT1A1, bovine LPO ovine PHS-1 liver cytosol (NQO1) NAT2) SULT1A2) rat and human rat and human NO2 NHOH liver microsomes NH2 liver microsomes (POR) (CYP1A1, CYP1A2) in vivo in rats/mice in vivo in rats/mice O O O 3-NBA N-OH-ABA 3-ABA Fig. 1. Pathways of metabolic activation and DNA adduct formation of 3-nitrobenzanthrone and 3-aminobenzanthrone; see text for details. R = -COCH3 or -SO3H; dA-N6-ABA, 2-(2'-deoxyadenosin-N6-yl)-3-aminobenzanthrone; dG-N2-ABA, N-(2'-deoxyguanosin- N2-yl)-3-aminobenzanthrone; dG-C8-N-ABA, N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone s277 Chem. Listy 101, s73−s310 (2007) 12. mezioborová česko-slovenská toxikologická konference a b 400 300 mAU WVL:254 nm WVL:254 nm mAU 3 - 24.252 300 1 - 4.767 phenacetine phenacetine 3-NBA 200 3 - 23.084 1 - 4.902 3-NBA 200 3-ABA 3-ABA 100 100 2 - 7.989 2 - 7.763 min min -50 -50 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 Fig. 2. HPLC of incubations of 3-NBA with NQO1 (a) and NADPH:cytochrome P450 reductase (b) a b 200 300 mAU WVL:254 nm mAU W VL:254 nm 1 - 4.921 1 - 4.783 150 phenacetine 200 phenacetine 2-NBA 2-NBA 100 100 50 2 - 24.258 3 - 25.422 min min -50 -20 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 Fig. 3. HPLC of incubations of 2-NBA with NQO1 (a) and NADPH:cytochrome P450 reductase (b) 10 3 -A B A 3 -N B A duction (fig. 4), dependence of converted 3-NBA and pro- duced 3-ABA on NQO1 (fig. 5) and 3-NBA concentra- Amount of transformed 3-NBA and gnerated 3-ABA [nmol] 8 6 tions (fig. 6). A time-dependent decrease in 3-NBA con- 4 centrations in incubations correlated with an increase in 3- 2 ABA formation (fig. 4). Reduction of 3-NBA to 3-ABA by 0 human NQO1 in the presence of NADPH exhibits the 10 20 30 40 T im e o f in c u b a tio n [m in ] 50 60 Michaelis-Menten kinetics (data not shown). The values of Michaelis constant Km of human NQO1, measured for 3- Fig. 4. Time-dependence of 3-NBA reduction by NQO1 NBA reduction and 3-ABA production, were 10.3 and 7.6 µM, respectively. In order to examine the molecular basis of the potent reduction of 3-NBA by human NQO1, the binding of 3- residues dissolved in 30 µl of methanol, and subjected to NBA to the active centre of NQO1 was modeled (fig. 7). RP-HPLC to evaluate the amounts of products of 3-NBA The model structures for the human NQO1-3-NBA- and 2-NBA reduction. HPLC was under isocratic condi- complex were calculated. It is evident that 3-NBA fits well tions of 70 % methanol, with a flow rate of 0.6 ml min−1. into the active site of human and rat NQO1, being bound The modeling of the binding of 3-NBA to the active site of near the isoalloxazine ring of the flavin prosthetic group of NQO1 was performed with the program Autodock 3.0.3. the enzyme. This allows an electron transfer during the and Sybyl 6.6.5 (Tripos GmbH, Germany) by the proce- reduction of 3-NBA. The value of the apparent dissocia- dure described7. tion constant for the human NQO1-3-NBA-complex was calculated to be 0.26 µM, respectively. Results and discussion Human NQO1 metabolizes 3-NBA to one major 10 3 -A B A product identified as its reductive metabolite found in 3 -N B A 8 urine samples of humans exposed to diesel emissions3, 3- Amount of transformed 3-NBA 6 and generated 3-ABA [nmol] ABA. Both compounds were separated by HPLC as two 4 distinguish peaks (fig. 2). NQO1 is the more effective enzyme to form this product than NADPH:CYP reductase (fig. 2a,b). In contrast to 3-NBA, no 2-NBA metabolites were generated by both enzymatic systems, which indicate 0 0 10 20 30 40 100 that this compound is a much worse substrate (if any) for C o n c e n t r a t io n o f N Q O 1 [µ g /m l] these enzymes than 3-NBA (fig. 3a,b). The metabolism of 3-NBA by human recombinant NQO1 was studied in de- Fig. 5. Dependence of 3-NBA reduction by NQO1 on the en- tail. We investigated the time-dependence of 3-NBA re- zyme concentration s278 Chem. Listy 101, s73−s310 (2007) 12. mezioborová česko-slovenská toxikologická konference 8 3 -A B A 7 3 -N B A Amount of transformed 3-NBA and generated 3-ABA [nmol] 6 5 4 3 1 0 0 5 10 15 20 50 C o n c e n tr a tio n o f 3 -N B A [µ M ] Fig. 6. Dependence of 3-NBA reduction by NQO1 on 3-NBA Fig. 7. 3-NBA docked to the active site of human NQO1 concentration Conclusion REFERENCES The results demonstrate for the first time that 3-NBA 1. Arlt V. M.: Mutagenesis 20, 399 (2005). is reduced by NQO1 and NADPH:CYP reductase to 3- 2. Nagy E., Johansson C., Zeisig M., Möller L.: J. Chro- ABA. The results also explain a strong mutagenicity and matogr., B 827, 94 (2005). carcinogenicity of 3-NBA in contrast to 2-NBA. Much 3. Seidel A., Dahmann D., Krekeler H., Jacob J.: Int. J. higher mutagenicity and carcinogenicity of 3-NBA evolve Hyg. Environ. Health 204, 333 (2002). from its potential to be easily reduced to reactive species 4. Nagy E., Zeisig M., Kawamura K., Hisumatsu Y., binding to DNA. Sugeta A., Adachi S., Moller L.: Carcinogenesis 26, 1821 (2005). Supported by grants 303/05/2195, MSM 0021620808 5. Arlt V. M., Bieler C. A., Mier W., Wiessler M., and 1M4635608802. Schmeiser H. H.: Int. J. Cancer 93, 450 (2001). 6. Arlt V. M., Stiborova M., Hewer A., Schmeiser H. H., Abbreviations Phillips D. H.: Cancer Res. 63, 2752 (2003). 7. Arlt V. M., Stiborova M., Henderson C. .J., Osborne CYP cytochrome P450 M. R., Bieler C.A., Frei E., Martinek V., Sopko B., NQO1 NAD(P)H:quinone oxidoreductase Wolf C. R., Schmeiser H. H., Phillips D. H.: Cancer NATs N,O-acetyltransferases Res. 65, 2644 (2005). SULTs sulfotransferases N-OH-ABA N-hydroxy-3-aminobenzanthrone s279


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Smock discusses the "new American family" on NPR
Pfeffer and colleagues re-examine impacts of community college attendance
Frey explains the minority-majority remapping of America
Apply for 2-year NICHD Postdoctoral Fellowships that begin September 2015
PSC Fall 2014 Newsletter now available
Martha Bailey and Nicolas Duquette win Cole Prize for article on War on Poverty
Michigan's graduate sociology program tied for 4th with Stanford in USN&WR rankings
Monday, Dec 1
Linda Waite, Health & Well-Being of Adults over 60
a PSC In The News reference, 2011
"Economists Express Caution on Forecasts in a Fed Study " - New York Times. 01/10/2011.
Matthew Shapiro says the Fed's plan to buy $600 billion in treasury securities might have a psychological basis. The Fed is sending a signal "that they're doing what they can and not throwing in the towel," he says.


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State Seeks To Prevent Falls By Older Ohioans
A state initiative to prevent falls by older Ohioans is aiming to warn shoppers of all ages what to look for and how to avoid falling hazards amid holiday shopping.
Despite the city's unemployment rate being the lowest in the state, Columbus custodians and security officers say they remain at near poverty levels. WOSU attended a picket Monday afternoon where workers called for better benefits.
Ohioans who repeatedly use guns to commit crimes would face tougher penalties under a proposed new law backed by Ohio Attorney General Mike DeWine and fellow Republicans.
The lawyer for a gay teacher fighting to get her job back at Bishop Watterson High School after her mother's obituary appeared in a newspaper with the name of her partner says Carla Hale will file a complaint with the City of Columbus under its anti-discrimination law.
City of Columbus police and fire unions contest new GPS tracking techonolgy being put into marked cars. The two unions, which represent about 3,000 employees, have asked city officials to re-open labor contract talks.
A state spokesman says a Columbus man was killed and a woman seriously injured in separate falls at southern Ohio's Hocking Hills State Park.


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Product DetailsHere is a great retro NFL shirt by Reebok.Made in 100% Polyester this shirt has cut and sewn panneling, contrast ribbed neckline,applique detail and badge detail.
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6 Minutes To Skinny ReviewWritten on November 15, 2014 at 8:52 am, by admin
So many people who are overweight want to lose weight but they want to do it really fast. There are some new diets and workouts that promise quick results and 6 Minutes To Skinny is one of these new approaches to shifting fat and shedding pounds in a hurry. There have been quite a few reviews of this weight loss program. Apparently it was developed by a well known nutritionist called Craig Ballantyne who knows a thing or two about getting into good shape. If you've seen his photos with his ripped abs and toned muscles then you'll realise that he's the right guy to ask for advice on how to get into shape. He is also a regular contributor to a number of fitness and weight loss blogs so he is seen as somewhat of an authority on the subject.
The "6 Minutes To Skinny" weight loss program is designed for both men and women and is supposed to leave you feeling really full of energy and not lethargic like some diets often do. There are some other diets live the Venus Factor which are designed just for women that have become so popular that they have even been translated into Spanish like Sistema Venus and others. Craig Ballantyne's program works for both sexes and as it becomes more popular will probably also come out in a Spanish version soon. The workouts that he has compiled all take less than 6 minutes and the foods he suggests eating promote weight loss but will still leave you feeling satisfied without all the cravings. A lot of the program is designed around giving the right hormones a boost and regulating their production to get your body into the fat burning mode you want to be in. Why not read the 6 Minutes to Skinny review and see if the program helps you get back into shape as quickly as you would like.
Buying Engagement Rings in South AfricaWritten on October 17, 2014 at 2:36 pm, by admin
When a woman plans her wedding she does this a long time before the day comes. She puts a lot of effort into thinking about exactly how she wants the day to happen. Of course she wants to look her best and so she starts to think about her wedding dress as well as her hair and makeup. Most of the things that will be seen at the wedding will never be seen again. There is one thing she will keep with her from that day and that item is her wedding and engagement ring. These are also things she would have thought a lot about.
Hopefully her fiance gave it a lot of attention and made a good choice. Choosing the best engagement rings can be a difficult task, especially for a man. It can be difficult to know what a good price is to pay and what design she would like. It is always a good idea for a man to do a lot of research online before buying the ring. If you would like to save some money and buy a good quality ring it would be a good idea to buy the engagement rings in South Africa. There are some very good online resources he can consult that give advice on how to buy engagement rings and wedding rings. These guides will help you become familiar with different designs. You will also learn about diamonds and how to choose the best one. The different kinds of precious metals that are available can also make the decision a challenge. Try to find out which colour gold your fiance prefers. It would be good to show her some pictures and have her show you which designs she likes.
You can try and keep it a secret but she probably already knows. Make sure that you choose the right engagement ring design so that she is happy to wear the ring forever. It would be good if she feels that she made a good choice in marrying you too.
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Song and Dance about Diets like The Venus Factor and Saffron ExtractWritten on May 6, 2014 at 1:06 pm, by admin
So many people today prioritize investing wisely in order to build up their wealth but far too little emphasis is placed on investing in our health. Examples like Steve Jobs, who had billions of dollars but sadly died of cancer, really emphasize that you can't put a price on your health. Those who do concern themselves with health all to often focus purely on weight loss and make a big deal about the latest diets on Dr Oz without really checking them out. This is hardly surprising as obesity has become one of the first world's biggest problems. Never before have we had the rate of both childhood and adult obesity that we see today. Staying in shape though is a result of healthy living and not just of the latest fad diet. This is really evident when we look at the lifestyles of indigenous people and their overall health.
Whether it's the native American Indians, descendants of the Maya in South America or the Maori's of New Zealand these cultures all seem to be associated with healthy, in-shape people. A lot of this can be attributed to their diet but also these people historically had to expend a lot of energy to get their food. Exercise was not a pass time for them but a necessity. A common component of indigenous cultures is music and dance. Dance has always formed an important part of their culture but it also promoted good health. The music and dancing had both a physiological as well as spiritual, or mental, benefit. When it came to weight loss and staying in shape these people also had the benefit of knowing about natural remedies such as Saffron Extract as well as others. Saffron Extract has been used for ages as a natural remedy for all sorts of issues from depression to weight loss. Saffron Extract has recently been featured by a number of health practitioners as a natural remedy and a number of suppliers have begun to sell these in tablet form as a diet supplement. An example of this is the well known Satiereal Saffron Extract.
With any diet our ultimate goal should always be to promote good health for both body and mind – not just weight loss. Diets like The Venus Factor Diet combine well designed movements with proper eating plans that aim to promote not just weight loss but have overall wellness as a goal. The Venus Factor system is a diet that focuses on not necessarily become extremely skinny but rather achieving the optimal shape for your body type. The Venus Factor comprises a set of well designed exercises along with an eating plan that is more than just about reduced calorie intake. If you're asking does the venus factor work then read some of the reviews on the web.
Being healthy is a state of mind. It's about lifestyle and attitude and not just about reducing calories or taking a pill. It's important to take the natural approach to weight loss. Listen to good music, dance more, move around, eat good food that's good for you and if you're going to take supplements make sure they're from nature's dispensary and not a lab.
Click here to read Melissa Schlag's Venus Factor Review


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Whether you are looking for a job in London or Loch Lomond, visit Jobs Today and start searching for the latest UK local jobs.
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Opinion: Healthcare savings without Medicare cuts
As President Barack Obama and congressional leaders continue discussions to avert the so-called fiscal cliff, we hear a troubling but familiar refrain in Washington: To fix our deficit, we must cut Medicare benefits. That is flat-out wrong. We have a systemwide health care cost problem in America. Health care expenditures are nearly 18 percent of our gross domestic product. The next least-efficient developed country in the world spends 12 percent of its GDP on health care. Leaders of both parties have acknowledged this dilemma. As Republican vice presidential nominee and House Budget Committee Chairman Paul Ryan has said, "If you want to be honest with the fiscal problem and the debt, it really is a health care problem."
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