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Evaluation of the SOS/umu-test post-treatment assay for the detection of genotoxic activities of pure compounds and complex environmental mixtures

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Evaluation of the SOS/umu-test post-treatment assay for the detection of genotoxic activities of pure compounds and complex environmental mixtures
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  Ž . Mutation Research 466 2000 161–171www.elsevier.com r locate r gentoxCommunity address: www.elsevier.com r locate r mutres Evaluation of the SOS r umu -test post-treatment assay for thedetection of genotoxic activities of pure compounds and complexenvironmental mixtures Bojan Hamer  a, ) , Nevenka Bihari  a , Georg Reifferscheid b , Rudolf K. Zahn b ,W.E.G. Muller  c , Renato Batel a ¨ a  Laboratory for Marine Molecular Toxicology, Center for Marine Research, Ru L  er Bosko Õ ic Institute, HR-52210 Ro Õ inj, Croatia ˘ ´ b  AMMUG, Commission for Molecular Biology, Academy of Sciences and Literature, D-55101 Mainz, Germany c  Department for Applied Molecular Biology, Institute for Physiological Chemistry, Johannes Gutenberg Uni Õ ersity, D-55099 Mainz, Germany Received 12 October 1999; received in revised form 27 January 2000; accepted 1 February 2000 Abstract This study presents an evaluation of the SOS r umu -test after introducing an additional dilution and incubation in thepost-treatment assay. This treatment reduces the influence of coloured test compounds that otherwise affect the colorimetricdetermination of the  b -galactosidase activity and the bacterial growth measurement during the testing of complexenvironmental samples. The post-treatment assay significantly increased the  b -galactosidase activity and consequently theenzyme induction ratios at higher doses of model genotoxins 4-nitroquinoline-  N  -oxide,  N  -methyl-  N  X -nitro-  N  -nitrosoguani- Ž . dine, 2-aminoanthracene, benzo  a  pyrene with low or no effect on the sensitivity of the test itself. On the other hand tests of environmental extracts indicated significant increases in sensitivity after additional incubation. 4-Nitroquinoline-  N  -oxidetreatments of bacteria in the test affected cell division and caused filamentous growth. The size of filamentous bacteria andincidence rate of the length categories was positively correlated with the concentrations of genotoxins. Presence of filamentous tester bacteria proved induction of SOS response and genotoxic activity of environment samples in SOS r umu -test.  q 2000 Published by Elsevier Science B.V. All rights reserved. Keywords:  SOS r umu- test; Post-treatment assay;  Salmonella typhimurium ; SOS response; Genotoxicity assay; Filamentous bacteria;Environmental pollution 1. Introduction Genotoxic chemicals can damage the genetic ma-terial of humans as well as that of organisms living ) Corresponding author. Tel.:  q 385-52-811-544; fax:  q 385-52-813-496. Ž .  E-mail address:  hamer@cim.irb.hr B. Hamer . in polluted environments. From the point of view of risk management, the potential effects of increasedfrequencies of DNA damage or mutations have to be w x considered 1 . Besides, any uncontrolled presence of genotoxins in any compartment of the natural envi-ronment is an unwanted situation. Early warningsystems of environmental hazards have become in-creasingly important in recent years. A number of  1383-5718 r 00 r $ - see front matter q  2000 Published by Elsevier Science B.V. All rights reserved. Ž . PII: S1383-5718 00 00016-4  ( ) B. Hamer et al. r  Mutation Research 466 2000 161–171 162 short-term bacterial test systems for determining bio-logical activities of genotoxins have been reported,among them the SOS r umu -test system offers some w x remarkable advantages 2–5 .The SOS r umu -test has been used for the determi-nation of the genotoxic potential of water andwastewater. For this purpose the test has been stan- w x dardized according to DIN and ISO 6,7 . The sys-tem is based on the ability of genotoxins to induceexpression of the  umu C gene, one of the SOS genesresponsible for ‘‘error-prone repair’’ and involved inmutagenesis more than other known SOS genes in w x bacteria 8,9 . The tester strain  Salmonella ty -  phimurium  TA1535 r pSK1002 carries a fused umu C– lac Z gene, allowing for the monitoring of  umu C expression by measuring  b -galactosidase ac-tivity in a colorimetric assay.The SOS r umu -test using a single tester strain w x detects many types of DNA-damaging agents 10,11 ,and can be used for the screening of samples con-taining amino acids and nutrients such as urine,serum and food compounds. Unsterile test materialcan be applied, since test results are available within5–6 h. The SOS r umu -test is potentially useful forthe detection of genotoxicity of complex environ- w x mental mixtures 12,13 and of airborne particles w x 14 . It can also be used for testing of compoundsthat have killing effects on tester bacteria, such as w x disinfectants 15 .Some strong yellow coloured extracts of environ-mental mixtures may affect the colorimetric determi- Ž . nation of   b -galactosidase activity E420 and estima- Ž . tions of bacterial growth E600 . To avoid theseproblems some authors incorporate a washing step of bacteria with TGA medium after compound incuba- w x tion 16,17 , or a post-treatment dilution and addi- w x tional reincubation for 2 h 12,18,19 . Post-treatmentassay may increase the sensitivity of the SOS r umu -test, a fact that can be used in the case of colouredenvironmental samples and in case of complex mix-tures, where the overall genotoxic effect might possi-bly be different from the sum of the effects of thesingle components.Post-treatment dilution and incubation have beenfirst described for the SOS chromotest in order toreduce effects of the yellowish colour of several test w x chemicals on the colorimetric assay 20 . Post-treat-ment dilution and incubation not only reduce thecolour of the test chemicals, but also gave higher b -galactosidase activity and enzyme induction ratios Ž .  w x IR 12 . The authors suggested that the reason forincreased IR during post-incubation may be due toan increase in the sensitivity of treated bacterial cellsto the remaining genotoxine or to the persistence of the SOS response.A post-treatment dilution after the first incubationperiod in the SOS r umu -test greatly decreases bacte-rial growth at higher concentrations of genotoxins,most of bacteria stop division and continue withfilamentous growth. The normal size of   S. ty -  phimurium  TA1535 r pSK1002 is 2–5  m m during thenormal cell cycle. The completion of the replicationcycle is connected with growth and division of thecell. There are also two cell division inhibitors SulA Ž . s SfiA and SfiC, both under the control of the w x SOS response sensitive to DNA damage 21,9 . Longfilaments are formed when septum formation is in-hibited, during perturbed DNA replication, growth w x and division 22,23 .This study was performed to evaluate the post- Ž . treatment assay dilution and incubation in theSOS r umu -test, with special consideration of fila-menotous bacterial growth, and its effect on  b -galactosidase activities and enzyme induction ratios. 2. Materials and methods 2.1. Chemicals Ž . Ž . Dimethylsulfoxide DMSO , benzo  a  pyrene Ž Ž . . Ž .  X B  a  P , 2-aminoanthracene 2-AA ,  N  -methyl-  N   - Ž . nitro-  N  -nitrosoguanidine MNNG , 4-nitroquinoline- Ž .  N  -oxide 4-NQO , amberlite XAD-7 were obtained Ž . from Sigma St. Louis, USA ; 2-mercaptoetanol and Ž . glutardialdehyde from Merck Darmstadt, Germany ; Ž bactotryptone from Difco Laboratories Detroit, . USA . Toluol, ampicilline, and  o -nitrophenyl- b - D - Ž . galactopyranoside ONPG were purchased from Ž . Serva Heidelberg, Germany . Commercial liver S9fraction from Aroclor 1254 induced rats was pur- Ž . chased from Organon Technika Belgium . The S9 w x mix was prepared according to Oda et al. 24 . Allother chemical reagents were of the highest commer-cial quality available.  ( ) B. Hamer et al. r  Mutation Research 466 2000 161–171  163 2.2. En Õ ironmental samples concentration and ex - traction Three sea water samples were taken from theRovinj region, one form uncontaminated control site Ž . island St. Ivan S1 , Rovinj harbour as polluted site Ž . S2 and waste water taken near the spill from Ž . Tobacco factory as an industrial pollution S3 . Sea- Ž . water 10 l. was extracted on amberlite XAD-7 resin Ž  3 . columns 20 cm for concentrating non-polar com-pounds. Before use, the resin was purified by ace-tone extractions, followed by several washing stepswith water. Flow rates of samples through thecolumns were forced by gravity and adjusted to 50ml r min. Adsorbed compounds were eluted with 100ml acetone, and the eluate was reduced nearly todryness by vacuum rotary evaporation. Subse-quently, the remaining solution was resuspended in Ž . DMSO 300  m l and stored at y 20 8 C prior to geno-toxicity assays. The final extract corresponding to 10l of extracted water was reduced to 300  m l, aconcentration factor of 1:33000. This means that 20 m l of extract corresponds to 666 equivalent volumes Ž . in ml of Sample EVmlS . 2.3. SOS  r umu-test  The SOS r umu -test was performed in test tubeswith and without S9 metabolic activation, with slight w x modifications according to Oda et al. 8 .  S. ty -  phimurium  TA1535 carrying a multicopy plasmidpSK1002 with fused  umu C- lac Z genes and the genefor resistance to ampicilline was used as the tester w x strain 25 . Monitoring of   umu C expression wasperformed by measuring the cellular ß-galactosidaseactivity, which reflects  umuC   induction. Theovernight culture was diluted 50-fold with fresh TGAmedium and incubated at 37 8 C until the bacteriareached log-phase bacterial growth. The incubationmixture consisted of the test compound dissolved in Ž . DMSO 20  m l or 20  m l of XAD-7 extracts, log-phase Ž . bacterial culture 800  m l and 0.1 M phosphate Ž . Ž . buffer pH 7.4 180  m l or S9 mix 180  m l contain-ing 4% of S9.All concentrations were tested in triplicate, witheach set of experiments usually repeated two or moretimes. After 2 h of exposure, the bacterial suspensionwas diluted 5-fold and 10-fold with warm TGAmedium, followed by a subsequent additional incuba-tion period of 2 h. At the end of treatment andpost-treatment incubation the bacterial growth was Ž . measured as turbidity E600 and the level of   b - Ž . galactosidase activity E420 was assayed by the w x colorimetric method using ONPG as a substrate 26 .The genotoxic activities were expressed in  b -galactosidase units and in enzyme induction ratios Ž . IR related to control samples treated with DMSO.Induction ratios above 1.5-fold are scored as suffi-cient positive results, estimated as minimal concen-trations of genotoxins required to produce statisti-cally significant increases from background controls Ž . according to Deutsches Institut fur Normung DIN ¨ and International Organization for Standardization Ž .  w x ISO standards 6,7 . Bacterial growth was ex-pressed in percent compared to the control. In allexperiments, the standard genotoxin 4-NQO was usedas positive control. The results of the tests are givenas mean of triplicate determination. SOS inductionratios after incubation and reincubation were testedfor significance level for the same concentration of genotoxins or EVmlS using Welsch multiple compa- w x ration test 27 . 2.4. Measuring and counting of bacteria by fluores - cence microscopy Subsequently to the standard SOS r umu -test, bac-terial samples from each incubation mixture werefixed with glutardialdehyde at a final concentrationof 1%, and stored at  q 4 8 C until work-up. Samples Ž . were filtered onto Nucleopore filters  f  0.2  m mstained previously with Irgeland black. The bacteriaon the filters were stained with acridine orange andanalysed with a Leitz Laborlux microscope using a100 =  Fluotar objective and Ploemopak epifluo-rescence equipment and a Leitz filter set providing a450–490 nm excitation band. Bacteria with greenfluorescence against a black background werecounted, measured in random fields at total magnifi- w x cation of 1000 =  28 . Bacteria were classified into Ž . seven length categories as: 1.0–5.6  m m class I , Ž . Ž . 5.7–11.2  m m class II , 11.3–16.8  m m class III , Ž . Ž . 16.9–22.4  m m class IV , 22.5–28.1  m m class V , Ž . 28.2–33.8  m m class VI and longer than 33.9  m m Ž . class VII .  ( ) B. Hamer et al. r  Mutation Research 466 2000 161–171 164Fig. 1. Effects of 4-NQO during incubation and additional post-treatment incubation in the SOS r umu -test on bacterial growth, b -galactosidase activity,  b -galactosidase units and enzyme induction ratios.  ( ) B. Hamer et al. r  Mutation Research 466 2000 161–171  165 3. Results and discussion Post-treatment 5-fold and 10-fold dilutions andadditional incubations were examined after exposureto different concentrations of 4-NQO. Five-fold dilu-tion and reincubation showed stronger increases of enzyme induction ratios compared to 10-fold dilution Ž . data not shown . Therefore, for all furtherSOS r umu -tests, a 5-fold dilution of the bacterialsuspensions was included after treatment incubation, Ž . Ž . Fig. 2. Bacteria  S. typhimurium  TA1535 r pSK1002 after incubation with a 20  m l DMSO and b 1.0  m g r ml 4-NQO in the SOS r umu -test.Bacteria were stained with acridine orange, and photos were taken under epifluorescence microscopy by total magnifications of 1000 times.Bar represents 5.6  m m.
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