Modyfikacje testu Salmonella do oceny mutagenności pyłowych zanieczyszczeń powietrza atmosferycznego

• Autorzy: Piekarska K.

Warianty tytułu

EN

Modified Salmonella assays for mutagenicity assessment of atmospheric dust pollutants

• Języki publikacji: PL

Abstrakty

PL

Celem pracy było wytypowanie procedury testu Salmonella, która pozwoliłaby wykrywać z dużą czułością obecność mutagennych zanieczyszczeń zaadsorbowanych na pyłach zawieszonych w powietrzu atmosferycznym aglomeracji miejskiej. Zwiększenie czułości testu jest niezwykle ważne, ponieważ w celu wykrycia aktywności mutagennej organicznych zanieczyszczeń powietrza należy pobrać znaczne jego objętości, co stanowi utrudnienie w wprowadzeniu testu do rutynowego monitoringu zanieczyszczeń powietrza. Praca obejmowała pobór próbek pyłu zawieszonego na terenie Wrocławia, jego ekstrakcję, analizę chromatograficzną i biologiczną. Badania prowadzono według klasycznego testu Salmonella, testu klasycznego bez udziału NADP jako składnika S9-mix, testu z 10-krotnie zwiększoną ilością bakterii, testu preinkubacyjnego prowadzonego w temperaturze 30 i 37 °C, testu w modyfikacji Kado oraz testu Mini, będącego kompilacją testu klasycznego i testu w modyfikacji Kado. Przeprowadzono porównanie przydatności frakcji mikrosomalnej aktywowanej standardowym induktorem, jakim jest bardzo niebezpieczny Aroclor 1254, z frakcją indukowaną fenobarbitalem oraz z frakcją mikrosomalną niepodaną uprzedniej indukcji. Rezultaty uzyskanych badań wykazały przydatność testu Salmonella w monitoringu zanieczyszczeń powietrza atmosferycznego aglomeracji miejskiej. Badane ekstrakty zanieczyszczeń powietrza różniły się między sobą sumaryczną zawartością oraz procentowym udziałem poszczególnych związków w zależności od miejsca i od sezonu poboru próbki. Zarówno sumaryczna zawartość WWA i nitroWWA w badanych próbkach, jak i ich spektrum było największe w okresie zimowym. Największą mutagennością odznaczały się próbki pobrane w zimie. Uzyskano duże współczynniki mutagenności w testach przeprowadzanych zarówno z aktywacją metaboliczną, jak i bez niej, świadczące o obecności promutagenów i mutagenów bezpośrednich. Mutageny zawarte w ekstraktach pyłów zawieszonych wywoływały silniejszą odpowiedź ze strony szczepów TA98 i YG1041, w porównaniu do szczepów TA100 i YG1042, co oznacza, że były to głównie związki wywołujące mutacje typu zmiany fazy odczytu. Największą przydatnością w wykrywaniu mutagenności ekstraktów pyłów zawieszonych odznaczał się szczep YG1041, szczególnie wrażliwy na nitrowe związki aromatyczne. Rezultaty badań wskazały na przydatność testu klasycznego przeprowadzanego z frakcją mikrosomalną indukowaną fenobarbitalem, bez wprowadzania do testu NADP w wykrywaniu promutagenów szczepem TA98. W przypadku szczepu YG1041 dobre wyniki uzyskano w teście klasycznym z 10-krotnie większą ilością bakterii bez udziału NADP. Dla obu tych szczepów wykazano także dużą przydatność testu Salmonella w modyfikacji Kado oraz testu preinkubacyjnego. Badania porównawcze frakcji mikrosomalnych stosowanych w testach wskazały, iż możliwe jest zastąpienie frakcji mikrosomalnej aktywowanej Aroclorem 1254 frakcją indukowaną fenobarbitalem oraz frakcją niepoddaną indukcji w badaniach nad mutagennymi zanieczyszczeniami powietrza aglomeracji miejskiej. Aroclor jest preparatem bardzo drogim, bardzo stabilnym chemicznie, który w śladowych ilościach powoduje mutacje wywołujące zmiany nowotworowe. Fenobarbital jest preparatem bezpieczniejszym i wielokrotnie tańszym. Wyniki badań wykazały konieczność każdorazowego doboru optymalnego stężenia frakcji mikrosomalnej wobec próbki środowiskowej, a nie wobec mutagenu standardowego. Należy także, prowadząc test z preinkubacją, każdorazowo, przed przystąpieniem do badań dobierać właściwy czas jej trwania. Wykazano bowiem, iż optymalny czas preinkubacji jest różny w zależności od rodzaju szczepu, temperatury preinkubacji i rodzaju frakcji mikrosomalnej.

EN

The aim of this work was to select such a procedure of the Salmonella assay that would lead to a highly sensitive detection of mutagenic contaminants adsorbed on dust particles suspended in the atmospheric air within urban agglomerations. Increased sensitivity of the assay is extremely important, as in order to detect a mutagenic activity of airborne organic pollutants, considerable air volumes have to be collected, which is a hindrance to introduction of the assay in a routine monitoring the atmospheric pollution. The study included: sampling suspended dust from the Wroclaw urban area, extraction of the dust collected, chromatographic and biological analyses. Investigations were based on a classical Salmonella assay, classical assay without NADP as an S9-mix component, assay using 10-fold higher bacteria count, preincubation assay at a the temperature of 30 °C and 37 °C, Kado-modified assay and the Mini assay - a sort of compiled classical assay and the Kado-modified assay. Suitability comparison was made between a microsomal fraction previously activated with a standard inductor such as the extremely dangerous Aroclor 1254, a phenobarbital-induced fraction, and a microsomal fraction, that had not been previously induced. The results obtained confirmed the Salmonella assay usability for the purpose of the atmospheric pollution monitoring within urban agglomeration. The extracts of atmospheric pollutants under examination differed in a total content and a percentage of individual compounds, depending on the place and the season of sampling. The highest total PAH content and the highest nitro-PAH content in the samples tested as well as the most extensive range of the compounds detected were found in winter season. The highest mutagenicity was noted for the samples collected in wintertime. High values of mutagenicity ratios were obtained from assays carried out both with and without metabolic activation, which testifies to the presence of both promutagens and direct mutagens. The mutagens contained in suspended dust extracts evoked a stronger response of the TA 98 and YG 1041 strains, compared to the TA 100 and YG 1042 strains, which means that to the most part those were compounds able to induce such mutations as the reading phase changes. The YG 1041 strain proved to be the most effective in the detection of mutagenicity of the suspended dust extracts, due to its notably high sensitivity to nitro-aromatic compounds. Ther results obtained from the investigations are indicative of the effectiveness of a classical assay performed by using a phenobarbital-induced microsomal fraction, with no NADP introduced in the assay for the detection of promutagens by the TA 98 strain. In the case of the YG 1041 strain, good results were obtained when performing a classical assay at 10-fold higher bacteria count, without NADP addition. The Salmonella assay as modified by Kado and the preincubation assay proved to be highly effective for both strains. Comparative examination of microsomal fractions used in assays has shown that the Aroclor 1254-activated microsomal fraction can be substituted by a phenobarbital-induced fraction or for a non-induced fraction in the investigations on mutagenic airborne pollutants in urban agglomerations. Aroclor is a very expensive preparation of excellent chemical stability, and its trace amounts cause cancer-linked mutations. Phenobarbital is a safe and far cheaper preparation. According to the study results, each time the optimal concentration of a microsomal fraction has to be selected in relation to an environmental sample, and not to a standard mutagen. Also, if an assay includes a preincubation step, each time before starting experiments, a suitable duration of the preincubation step has to be selected. It was proved namely that the optimum preincubation time would be different, depending on the type of a strain, preincubation temperature and the type of a microsomal fraction.

Słowa kluczowe

PL

frakcja PM10; ekstrakty pyłu zawieszonego; aglomeracja miejska; test Salmonella; test preinkubacyjny; test w modyfikacji Kado; test w formie Mini; NADP; Aroclor 1254; fenobarbital; Salmonella typhimurium TA 98; TA 100; YG 1041; YG 1042; zanieczyszczenia atmosfery

EN

NADP; Aroclor 1254; Salmonella typhimurium TA 98; TA 100; YG 1041; YG 1042; dust pollutants; PM10 fraction; Salmonella assay; urban agglomeration; phenobarbital; preincubation assay; atmospheric pollution

• Wydawca: Oficyna Wydawnicza Politechniki Wrocławskiej
• Czasopismo: Prace Naukowe Instytutu Inżynierii Ochrony Środowiska Politechniki Wrocławskiej. Monografie
• Rocznik: 2008
• Tom: Vol. 87, nr 52
• Strony: 196--196
• Opis fizyczny: s., bibliogr. 223 poz.

Twórcy

autor

Piekarska K.

• Zakład Biologii i Ekologii Instytutu Inżynierii Ochrony Środowiska Politechniki Wrocławskiej, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław

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