DNA content alterations in Tetrahymena pyriformis macronucleus after exposure to food preservatives

Acta Biologica Hungarica 63(4), pp. 483–489 (2012)DOI: 10.1556/ABiol.63.2012.4.70236-5383/$ 20.00 © 2012 Akadémiai Kiadó, BudapestDNA coNteNt AlterAtioNsiN TeTrAHymenA pyrIfOrmIs mAcroNucleus After exposure to fooD preserv Atives soDiumNitrAte AND soDium beNzoAteA riAdni C. L outsidou ,1 V AsiLiki i. H Atzi ,2 C. t. C HAsApis ,1G eorGiA i. t erzoudi ,2 C HArA A. s piLiopouLou 3 and M AriA e. s tefAnidou 3 *1 Department of pharmacy, university of patras, Gr-26504, patras, Greece2 institute of Nuclear & radiological sciences & technology, energy & safety, National centre forScientific Research, NCSR “Demokritos”, Ag. Paraskevi Attikis, Athens, Greece3 Department of forensic medicine and toxicology, medical school, university of Athens,75 M. Asias str., Goudi 115.27, Athens, Greece(received: November 29, 2011; accepted: January 23, 2012)the toxicity, in terms of changes in the DNA content, of two food preservatives, sodium nitrate and sodium benzoate was studied on the protozoan Tetrahymena pyriformis using DNA image analysis tech-nology. For this purpose, selected doses of both food additives were administered for 2 h to protozoa cultures and DNA image analysis of T. pyriformis nuclei was performed. The analysis was based on the measurement of the Mean Optical Density which represents the cellular DNA content. The results have shown that after exposure of the protozoan cultures to doses equivalent to ADI, a statistically significant increase in the macronuclear DNA content compared to the unexposed control samples was observed. The observed increase in the macronuclear DNA content is indicative of the stimulation of the mitotic process and the observed increase in moD, accompanied by a stimulation of the protozoan proliferation activity is in consistence with this assumption . since alterations at the DNA level such as DNA content and uncontrolled mitogenic stimulation have been linked with chemical carcinogenesis, the results of the present study add information on the toxicogenomic profile of the selected chemicals and may poten -tially lead to reconsideration of the excessive use of nitrates aiming to protect public health.Keywords: Tetrahymena pyriformis – sodium nitrate – sodium benzoate – DNA content – mean optical DensityiNtroDuctioNpreservatives are widely used in food, beverages, drugs and cosmetic industry during processing or storage to preserve flavour, to prolong the shelf-life of food and protect it against deterioration caused by microorganisms, to ensure its nutritional adequacy and safety or to enhance its taste and appearance. Among them, two of the most com -monly used food preservatives are sodium nitrate and sodium benzoate [11, 19, 27].sodium nitrate (NaNo 3, E251) due to its antimicrobial properties against C. botu-linum growth is widely used in the processing of red meat [11]. A small amount of* C orresponding author; e-mail: mstefan@med.uoa.gr484A riAdni C. L outsidou et al.Acta Biologica Hungarica 63, 2012the nitrate added to processed meat as a preservative is reduced to nitrite, in addition to any nitrite that may also be added. The nitrite then reacts with secondary amines to produce the potent carcinogens N-nitrosamines [33]. About 5% of nitrates con -sumed by healthy adults are reduced to nitrites by bacteria in saliva and alimentary tract. Sodium nitrate has been associated with disease conditions including reproduc -tive toxicity, anaemia and thyroid hypertrophy, while according to World cancer research fund uK, the addition of nitrates in the processed meat, which can be fur-ther metabolized to nitrites, has been linked with increased risk of colon cancer [6].sodium benzoate (Nac 6H 5co 2, e211) due to its bacteriostatic and fungistatic activities [2, 14, 26] is widely used as a food preservative in acidic foods, such as salad dressings, carbonated drinks, jams and fruit juices, pickles, purees and pie fill -ings [27]. It is also found in cough syrups and in alcohol-based mouthwash. Although the international program on chemical safety (ipcf) found no adverse effects in humans (at doses of 647–825 mg/kg of body weight per day) [3, 20], some studies have linked exposure to sodium benzoate with mild hypersensitivity reactions in human and experimental animals, such as allergic reactions [4], immune responses suppression [17], allergic contact dermatitis [32] inhibition of gluconeogenesis, ure-agenesis [7] and fatty acid oxidation [13].since the widely used food preservatives sodium nitrate and sodium benzoate have been accused for various toxic reactions such as hypersensitivity reactions (allergic reactions, immune responses suppression, allergic contact dermatitis), asthma, decreased lung function (chronic obstructory pulmonary disease) [1, 17, 20, 21], the aim of this study was to test their in vivo toxicity in terms of DNA content changes of the protozoan, Tetrahymena pyriformis (T. pyriformis ) by means of the computer-ized DNA image analysis system (CIAS), [22–24, 27–31, 38].Protozoa are eukaryotic cells widespread in the aquatic and terrestrial environ -ment. Their normal behaviour in nature may be related to the presence of pollutants and to air, soil and water quality. Among the various species of protozoa, ciliates are the most frequently used as experimental organisms [25, 36]. The protozoan T. pyri-formis is a ciliate monocellular organism and it is established as an alternative experimental model for functional, pharmacological, toxicological or immunotoxic studies [22, 28]. Our previous laboratory experience with Tetrahymena has led us to consider the DNA content (measured as mean optical Density), as a valuable param-eter to test responses to various toxicants. Tetrahymena contains one macronucleus, and many strains contain a micronucleus as well. The macronucleus of the protozoan contains 12–24 timesfold DNA in comparison to micronucleus, and practically repre-sents the total DNA content of the protozoan. Macro- and micronuclei both give strongly positive cytochemical reactions for DNA with Feulgen reagent [37]. Based on previous studies where DNA content has been proposed as a cytogenetic marker of toxicity for several chemical substances (butylated hydroxytoluene, tartrazine) and drugs (cocaine, crack) [29, 31], the aim of this study was to estimate the possible T. pyriformis macronuclear DNA content changes after exposure to selected doses of the food additives sodium nitrate and sodium benzoate by means of the DNA image technology.food additive toxicity on T. pyriformis macronuclei485Acta Biologica Hungarica 63, 2012mAteriAls AND metHoDs Culture conditions and chemical exposureT. pyriformis protozoa were grown axenically in a proteose-peptone broth at 25 °c, until reaching late log phase. The selected food preservatives (sodium nitrate and sodium benzoate) (sigma-Aldrich, Germany) were added separately to a 100-ml cul-ture as aqueous solutions. The selected doses were 4 × 10–4 M, 8 × 10–4 m (ADi) and 40 × 10–4 M for sodium nitrate and 2.5 × 10–4 M, 5 × 10–4 M (ADI) and 25 × 10–4 m for sodium benzoate. These doses were selected based on previous studies carried out in our lab, with the use of human lymphocytes [16], where the ADi doses for both food additives were the minimum effective doses. According to this study a spontaneous and stimulated DNA synthesis was observed. For each tested substance, an experi -mental series consisting of six cultures of the protozoan was used and another experimental series served as control. The cultures were allowed to stand for 2 hours after administration of food additives. Then, samples were obtained from each group of protozoan cultures and were observed under the microscope to check the viability, morphology and motility of the protozoa. All these parameters were normal. Afterwards, the protozoan population was counted using the hematocytometer under the microscope at a magnification of ×80 and Feulgen staining followed. Unexposed cultures of T. pyriformis were used as control samples.feulgen stainingOne drop (50 μl) of each protozoa culture, was pipetted onto a microscope slide, impregnated with the adhesive agent poly-l-lysine hydrobromide (sigma-Aldrich, Germany) 0.1% in water for injection, and was allowed to stand until drying [12]. The DNA of the protozoa nuclei was stained red to purple with the feulgen reagent to stain DNA specifically and quantitatively, according to the static cytophotometric procedure [29, 31].DnA image analysisDNA image analysis technique is based on a conversion of optical images of cells into arbitrary numerical values (digitization) based on light absorbance or transmis-sion and has proved to be an effective approach to examine and classify stained cells spread on a glass slide in the form of smears [29]. The counting of macronuclei and the mean optical density [18] of T. pyriformis macronuclei was determined by means of the digital DNA image analysis system (CIAS, SAMBA 2000) [16]. A total number of 50 T. pyriformis macronuclei from each culture were selected randomly from each sample (i.e. microscopic slide) and the quantification of MOD was performed at the wavelength of 560 nm. The statistical analysis of the results was carried out using the486A riAdni C. L outsidou et al.Acta Biologica Hungarica 63, 2012student’s t -test and mean values and standard deviations were calculated from six independent experiments. All p -values were considered statistically significant at p < 0.05.results AND DiscussioNin the present study, using DNA image analysis system, the DNA content (expressed as mean optical Density, moD) of T. pyriformis macronuclei after exposure to sodium nitrate and sodium benzoate has been estimated. The results have shown that both sodium nitrate and sodium benzoate (at the ADi value) affect the DNA content of T. pyriformis macronuclei when compared to the control samples (Table 1). In particular, the observed statistically significant increase in MOD after exposure to sodium benzoate and sodium nitrate was from 29.69 ± 8.49 (control value) to 33.99 ± 7.31 and 40.43 ± 8.43 (t = 6.34, p < 0.001) (t = 2.54, p < 0.01) respectively. Smaller doses from ADI have shown no statistically significant difference of the two parameters (moD and the population of the protozoa) in comparison with the control samples, while the larger doses selected had influenced significantly the protozoan viability and caused protozoan cell death.the observed increase in the macronuclear DNA content as a result of ADi values of both sodium nitrate and sodium benzoate is probably the consequence of the pre-servative-induced stimulation of the macronuclear DNA synthesis which leads to an increase in the mitogenic activity of protozoa. In particular, the preservative thatTable 1mean optical Density of the DNA content of 50 macronuclei of the protozoan T. pyriformis after 2 hoursexposure to sodium nitrate and sodium benzoatepreservativeconcentrationprotozoa/mlMOD ± SDcontrol – 2.4 × 10529.69 ± 8.49sodium nitrate4 × 10–4 m 2.6 × 10531.63 ± 7.01(p < 0.05)8 × 10–4 m (ADi) 4.7 × 10540.43 ± 8.43(t = 6.35, p < 0.001)40 × 10–4 m––sodium benzoate 2.5 × 10–4 m 2.1 × 10528.21 ± 9.54(p < 0.05)5 × 10–4 m (ADi) 3.3 × 10533.99 ± 7.31(t = 2.54, p < 0.01)25 × 10–4 m––the statistical analysis of the results was carried out using the student’s t -test and mean values and standard deviations were calculated from six independent experiments. All p -values were considered statistically significant at p < 0.05.food additive toxicity on T. pyriformis macronuclei487Acta Biologica Hungarica 63, 2012induces the highest increase in the protozoan moD value after exposure is also expected to induce the higher increase in the proliferative activity. Indeed, the observed statistically significant increase in the proliferative activity after exposure of T. pyriformis to sodium nitrate and sodium benzoate, with respect to controls, sup-ports this hypothesis. Specifically, a higher increase in cell division was observed in cultures treated with sodium nitrate [from 2.4 × 105 protozoa per ml (control value) to 4.7 × 105 cells/ml], than with sodium benzoate (3.3 × 105 cells/ml). Furthermore, sodium nitrate induces a higher increase in the moD value compared to sodium benzoate.in the present study, the increase in the macronuclear DNA content after exposure of T. pyriformis to sodium nitrate and sodium benzoate, results from a stimulation of the mitotic process which is expressed here as an increase in cell division. In general, it is well known that cytogenetic changes at the DNA level [8, 35], as well as uncon -trolled cellular proliferation [5, 9, 10, 15] are causative factors of genetic instability and carcinogenesis, while DNA content (DNA index) has been widely used in many studies as a significant prognostic tool of aneuploid tumors [34, 35, 38]. Therefore, the observed above-normal increase of the DNA content and cellular division pro-cesses after exposure to both food additives might indicate their potential carcino-genic profile. Moreover, similar cellular alterations might act antagonistically in other cellular processes of the living cells. For example, the energy deposit of the proto -zoan cell is consumed in the chemically-induced mitotic processes induction and other life-supporting mechanisms, such as phagocytosis, that is highly suppressed [28, 29]. However, further studies on the impact of both food preservative in other morphonuclear and densitometric parameters (e.g. area, ploidy balance, degree of aneuploidy), will elucidate whether the observed chemically induced increase in DNA content may lead to disturbances in the T. pyriformis chromosomal segregation during cellular division that would potentially lead to aneuploidy.In conclusion, the findings of the present work support a toxicogenomic effect of both sodium nitrate and sodium benzoate in the selected experimental model, T. pyri-formis which is expressed as an increase in the DNA content and protozoan prolif-erative activity. 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