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Effects of prometryne on apoptosis and necrosis in thymus, lymph node and spleen in mice
Environmental Toxicology and Pharmacology 27 (2009) 182–186
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Effects of prometryne on apoptosis and necrosis in thymus, lymph node and spleen in mice Domagoj Ðikic´ a,∗ , Snjeˇzana Zˇ idovec-Lepej b , Anica Remenar b , Kreˇso Bendelja c , Vesna Benkovic´ a , Anica Horvat-Kneˇzevic´ a , Gordana Brozovic´ d , Nada Orˇsolic´ a a
Faculty of Science, University of Zagreb, Department of Animal Physiology, Biology Division, Rooseveltov trg 6, HR-10000 Zagreb, Croatia Molecular Diagnostics Laboratory of University Hospital for Infectious Diseases “Dr. Fran Mihaljevi´c”, Mirogojska 8, 10000 Zagreb, Croatia Institute of Immunology, Rockefellerova 2, 10000 Zagreb, Croatia d University Hospital for Tumors, Ilica 197, HR-10000 Zagreb, Croatia b c
a r t i c l e
i n f o
Article history: Received 15 July 2008 Received in revised form 28 September 2008 Accepted 4 October 2008 Available online 15 October 2008 Keywords: Prometryne Immunotoxicity CBA mice Triazine Flow cytometry
a b s t r a c t Prometryne is a methylthio-s-triazine herbicide. Significant traces are documented in environment, mainly waters, soil and plants used for nutrition. The aim of this study was to estimate prometryne immunotoxic properties through induction of apoptotic and/or necrotic changes in thymocytes, splenocytes and lymph node cells after repeated subchronical exposure. Three different doses of prometryne (185, 375, 555 mg kg−1 ) were applied per os every 48 h, over 28 days. Flow cytometry assay (annexinVFITC and PI) was conducted to record apoptotic and necrotic damage. In the spleen significant changes in the percentage of apoptotic cells were not detected between treated and control groups respectively. In thymus and lymph node, within the lowest dose group (185 mg kg− 1), an increase in percentage of early apoptosis without any significant increase in necrosis was detected. Medium (375 mg kg−1 ) as well as high dose triggered increase in late apoptosis in lymph node while in thymus; late apoptosis was increased only in animals exposed to the highest dose (555 mg kg−1 ). The highest applied dose, in thymus and lymph node respectively, caused a general decrease in percentage of vital cells in favour of marked increase of percentages of all types of dying cells (apoptotic, late apoptotic/early necrotic and necrotic). Prometryne caused disbalance in major organs of immune system, markedly lymph nodes and thymus, by induction of early apoptotic changes in dose/time specific manner. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The use of 1,3,5-triazine or s-triazine herbicides in agriculture has increased the levels of xenobiotic compounds in soil, water and air, and present a serious risk to the environment and human health for over three decades (Brusick, 1994). Herbicide prometryne belongs to a group of methyl-thio-s-triazines and its molecular structure is similar to renowned atrazine, differing only in sulfur instead of chlorine (Kamrin and Montgomery, 2000; LeBaron et al., 2008). Both herbicides possess similar toxicokinetic properties within mammalian organism (Adams et al., 1990; Maynard et al., 1999). Herbicide atrazine belongs to a group of chloro-striazines and different toxic effects such as genotoxicity, mutagenic, endocrine disrupting and immunotoxic properties were confirmed (Boobis et al., 2008), which finally resulted in its restricted use or complete ban in many countries (Pino et al., 1988; Dunkelberg
∗ Corresponding author. Tel.: +385 1 48 777 47/53; fax: +385 1 48 26 260. ´ E-mail address: magistar [email protected] (D. Ðikic). 1382-6689/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2008.10.002
et al., 1994; Ribas et al., 1995; Lioui et al., 1998; Gammon et al., 2005). Contrary to atrazine, only slight information on toxic effects of prometryne and other methylthio-s-triazine has been published, presumably for a belief that it is a moderately toxic pesticide related to its relatively high LD50 for mice = 3750 mg kg−1 (EPA-RED, 1996). Majority of experiments investigating toxicity of prometryne were conducted in vitro or ex vivo on experimental models such as bacteria and invertebrates where prometryne was not applied alone but rather often formulated in mixtures with other chemicals (Egert and Greim, 1976; Rozek, 1978; Kaya et al., 2000; Kurebayashi, 2005). Prometryne or 1,3,5-triazine-2,4-diamine(N,N -bis(1methylethyl)-6-(methytio) is used for the control of annual broadleaf and grass weeds in crops corn, carrots, parsley, peanuts, cotton, pigeon peas, alfa alfa, and many other cultivated plants. Prometryne and residues of prometryne can be measured in significant concentrations especially in rural areas and third world countries where its use is in considerable quantities (EPA-RED, 1996; Kamrin and Montgomery, 2000; EPA IRIS, 2001; AmadorRamírez et al., 2007; Tunku et al., 2007). It is relatively persistent in waters (Berg et al., 1995; Leh et al., 2005; Hua et al., 2006)
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and soil (Fiveland, 1977; Muller and Britz, 1982) or even in air near production or application sites (Schumann et al., 1990). Significant traces are also documented in plants used as human and domestic animal nutrition (Fiveland, 1977; Dubenetskaia et al., 1981; Bardalaye and Wheeler, 1985; Zhang et al., 2006) and some medical plants protected with herbicide prometryne (Reifenstein and Pank, 1975). Beside that traces of prometryne could be found in cow and breast milk (Krivankova et al., 1989; Balduini et al., 2003). Some investigations point to immunohematotoxicity and suppressed cellular immune response (Gzhegotskii, 1968; Kulakov, 1970; Giurgea et al., 1981). It is established that the prometryne induce DNA fragmentation, a hallmark of apoptosis in blood leukocytes (Ðikic´ et al., 2009). Therefore, this study was designed to evaluate the apoptotic and/or necrotic induction potential of prometryne in thymocytes, splenocytes and lymph node cells after repeated in vivo subchronical exposure to three different doses on mice model. Flow cytometry assay with annexinV-FITC and PI stain, as a highly sensitive method was selected to record early apoptotic and necrotic events. This technique is based on the observation that soon after initiating apoptosis, cells translocate the membrane phosphatidylserine (PS) from the inner face of the plasma membrane to the cell surface while necrotic cells become more permeable to propidium iodide. 2. Materials and methods 2.1. Animals Inbred CBA mice (65 ± 3 days old), from the mouse colony of Faculty of Science, University of Zagreb were used. The animals were maintained on a pellet diet (Pliva dd, Zagreb, Croatia) and water was provided ad libitum. The animals were maintained under 12L:12D hours light–dark regime. Experiments were carried out according to the guidelines force in Croatia (Law on the Welfare of Animals, NN # 19, 1999) and in compliance with the Guide for the Care and Use of Laboratory Animals, DHHS Publ. # (NIH) 86-123 and OECD guidelines for subchronic (28 days) toxicity testing in rodents (OECD, 1995). 2.2. Treatment of experimental animals with prometryne Mice where randomly distributed to groups of 10 individuals of both sexes. Within each group (N = 10, 5♀ + 5♂) mice were receiving low, medium and high doses of prometryne (C10 H19 N5 S), CAS No. 7287-19-6, EPA Reg. No. 9779-297 (tech. grade 95%, Herbos dd, Sisak, Croatia). Control group (N = 10, 5♀ + 5♂) received no prometryne. The prometryne doses were 185, 375 and 555 mg kg−1 respectively. The medium dose represents 1/10 of the LD50(mice) = 3750 mg kg−1 (EPA-RED, 1996). Dose of 185 mg kg−1 which is 1/20 of the LD50 and two fold lower than middle dose used, was selected as first effective dose (ED10 ) recognized through pilot tests. Dose of 555 mg kg−1 was selected as highest effective dose without lethal toxicity (LD10 ) during 28 days and also recognized through pilot tests. The metabolic study of Maynard et al. (1999) showed that the majority of the absorbed prometryne is metabolized or excreted within 48 h. To avoid overdose through bioaccumulation (log PKo/w = 3.5; Kamrin and Montgomery, 2000) mice were given prometryne per os, every 48 h, during 28 days in doses (185, 375 and 555 mg kg−1 ). Prometryne was prepared as a corn oil suspension, in volume of 0.2 ml per animal. Appropriate control group was selected and treated with the same volume of corn oil. The whole experiment was repeated three times. 2.3. Determination of body weight and organ Mice were weighed every 2 days during the experimental period starting on day 1 after application of prometryne p.o. The organs were collected on the 28th day of the experiment respectively, by protocols described in EMPReSS, SOP (Standard Operating Procedure (Green et al., 2005) but also see: http://empress.har.mrc.ac.uk) and processed to cell extractions. 2.4. Tissue cell preparation, cell staining and flow cytometry Single cell suspensions were prepared from spleen, thymus and popliteal lymph nodes of each treated animal by gently rubbing the organs through nylon mesh filter. Cellular debris was removed and cells were washed in PBS and resuspended (105 cells ml−1 ). For cell staining the standard Annexin-V-FITC Detection Kit (BioVision® ) was used according to manufacturer instructions. Briefly, 105 cells were
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resuspended in 100 L of binding buffer and 10 L of annexin-V-FITS (fluorescein isothiocyannate-labelled annexin V) and PI (propidium iodine) was added. After 15 min of incubation at 30 ◦ C, additional 400 L of binding buffer was added and reading was taken on flow cytometer FC 500 CYTOMICS Beckman Coulter, Florida, USA (EX = 488 nm, EM = 525 nm). The gate was same for both the exposed and control mice and the results are shown as percentage of positive cells within gate. Analysis was performed by CXP 2.0 software. 2.5. Statistical analysis Statistical analyses were performed using Statistica 5.0 software (StatSoft, Tulsa, USA). Each sample was characterized considering the mean (±standard deviation of the mean). The unit of measurement was the animal. Multiple comparisons between groups were done by means of ANOVA. Post hoc analysis of differences was conducted by Scheffé and Duncan test to establish the differences between groups. Level of statistical significance was set at p ≤ 0.05 and in Tables differences are marked according to LSD system.
3. Results The body weights of all experimental animals were not significantly changed during experiment (Table 1). Weight of spleen decreased significantly (p ≤ 0.05) in the group of animals receiving the highest dose (555 mg kg−1 ) while weight of thymus and lymph nodes were not significantly changed (Table 1). The percentage of unstained cells (annexinV−/PI−) was significantly decreased (p ≤ 0.05) in thymus (Table 2) and lymph node (Table 4) in the highest dose (555 mg kg−1 ) compared to the control. There was no significant difference in percentage of unstained cells in thymus (Table 2) and lymph node (Table 4) within the low (185 mg kg−1 ) and middle (375 mg kg−1 ) dose compared to the control. The percentage of unstained cells in spleen was unaffected by prometryne in any applied dose compared to control (Table 3). The percentage of cells in early apoptosis (annexinV+/PI−) was significantly increased (p ≤ 0.05) in thymus (Table 2) and lymph node (Table 4) treated with doses of 185 and 555 mg kg−1 compared to the control. while no differences were found in percentage of cells in spleen (Table 3). The percentage of cells in late apoptosis (annexinV+/PI+) was significantly increased (p ≤ 0.05) in thymus exposed to the highest dose (Table 2) compared to control animals. In lymph node significantly increased percentage of cells in late apoptosis was observed in medium and high dose groups compared to the control groups (Table 4). There were no significant changes in annexinV+/PI+ splenocytes in any of the dose groups compared to the control (Table 3).
Table 1 Body weight of mice throughout the experiment and organ in control group and groups of animals treated with three different doses of prometryne. Body weight1 (mean ± S.D.) 0 mg kg−1 Dose of prometryne 1st day (g) 21.55 ± 2.57 14th day (g) 21.57 ± 2.83 28th day (g) 21.65 ± 3.05
185 mg kg−1 21.83 ± 3.11 22.08 ± 2.88 22.40 ± 2.88
375 mg kg−1 21.47 ± 3.56 21.62 ± 3.16 21.40 ± 3.11
555 mg kg−1 21.57 ± 2.21 20.86 ± 2.17 21.83 ± 3.11
Organ weight1 (mean ± S.D.)
Dose of prometryne Thymus (mg) Spleen (mg) Lymph node (mg)
0 mg kg−1
185 mg kg−1
29.48 ± 3.91 86.24 ± 9.61b 10.25 ± 4.96
27.00 ± 7.90 25.92 ± 7.50 85.3 ± 10.2b 89.6 ± 10.0b 8.26 ± 3.49 8.30 ± 2.50
375 mg kg−1
555 mg kg−1 29.31 ± 3.34 75.7 ± 14.2a 11.16 ± 4.29
1 Body weight was measured every second day of the experiment, for simplicity only three measurements are shown. a,b Within row, means with different superscripts (letters) are significantly different (p ≤ 0.05).
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Table 2 Percentages of cells in thymus of control group and groups of animals treated with three different doses of prometryne. 0 mg kg−1
Doses of prometryne Unstained cells (annexinVFITC−/PI−) Early apoptosis (annexinVFITC+/PI−) Late apoptosis (annexinVFITC+/PI+) Necrosis (annexinVFITC−/PI+) a,b,c
185 mg kg−1
375 mg kg−1
555 mg kg−1
Mean ± S.D.
Min.
Max.
Mean ± S.D.
Min.
Max.
Mean ± S.D.
Min.
Max.
Mean ± S.D.
Min.
Max.
± ± ± ±
76.7 0.0 0.1 0.7
96.5 11.6 10.5 3.3
79.3 11.8 6.8 1.9
± ± ± ±
62.6 5.3 3.6 0.6
86.1 22.3 17.5 4.5
83.7 8.3 5.1 2.7
± ± ± ±
78.7 0.5 0.0 1.1
95.9 11.8 8.6 3.8
70.3 16.1 10.7 2.6
± ± ± ±
54.2 10.9 9.1 0.7
77.3 25.5 13.9 6.2
85.5 6.7 5.9 1.7
6.9b 4.1a 3.6a 0.9a
7.1b 5.4b 3.3a 1.0a
5.1b 3.5a 2.3a 0.9b
9.3a 5.9c 1.8b 2.1b
Within row, means with different superscripts (letters) are significantly different (p ≤ 0.05).
Table 3 Percentages of spleen cells of control group and groups of animals treated with three different doses of prometryne. 0 mg kg−1
Doses of prometryne Unstained cells (annexinVFITC−/PI−) Early apoptosis (annexinVFITC+/PI−) Late apoptosis (annexinVFITC+/PI+) Necrosis (annexinVFITC−/PI+) a,b
185 mg kg−1
375 mg kg−1
555 mg kg−1
Mean ± S.D.
Min.
Max.
Mean ± S.D.
Min.
Max.
Mean ± S.D.
Min.
Max.
Mean ± S.D.
Min.
Max.
± ± ± ±
60.0 1.7 2.4 2.8
86.7 13.4 14.8 7.0
78.1 6.6 9.0 6.0
± ± ± ±
69.6 3.3 4.6 3.0
82.1 10.6 15.9 10.3
79.1 5.0 9.6 5.7
± ± ± ±
77.5 2.2 7.8 5.1
81.9 7.4 11.1 6.4
76.6 8.8 10.7 3.7
± ± ± ±
71.9 6.0 6.1 2.7
85.0 10.9 15.4 5.0
77.1 6.6 8.7 5.1
7.9 2.8 3.7 2.2a
3.3 2.1 2.7 2.2a
1.7 1.8 1.2 0.5a
5.4 2.0 3.6 0.9b
Within row, means with different superscripts (letters) are significantly different (p ≤ 0.05).
Table 4 Percentages of lymph node cells of control group and groups of animals treated with three different doses of prometryne. 0 mg kg−1
Doses of prometryne Unstained cells (annexinVFITC−/PI−) Early apoptosis (annexinVFITC+/PI−) Late apoptosis (annexinVFITC+/PI+) Necrosis (annexinVFITC−/PI+) a,b,c
185 mg kg−1
375 mg kg−1
555 mg kg−1
Mean ± S.D.
Min.
Max.
Mean ± S.D.
Min.
Max.
Mean ± S.D.
Min.
Max.
Mean ± S.D.
Min.
Max.
± ± ± ±
77.4 1.4 1.0 0.3
94.6 14.7 19.4 1.6
83.2 10.7 5.3 0.7
± ± ± ±
64.9 5.9 2.2 0.3
88.4 15.6 6.4 1.5
77.7 7.3 13.6 1.2
± ± ± ±
71.0 6.8 9.1 0.7
82.2 7.9 20.2 1.6
69.4 13.5 15.3 1.6
± ± ± ±
58.1 12.1 9.0 0.5
75.7 14.8 25.3 2.6
83.0 8.8 7.3 0.8
7.3b 3.7a 4.6a 0.2a
3.3b 3.2b 1.2a 0.3a
5.9b 0.5a 5.8b 0.5b
7.2a 1.2b 6.3b 0.8b
Within row, means with different superscripts (letters) are significantly different (p ≤ 0.05).
The percentage of necrotic cells (annexinV−/PI+) in thymus (Table 2) and lymph node (Table 4) was significantly increased (p ≤ 0.05) in middle and high doses, while significant decrease (p ≤ 0.05) was observed in spleen (Table 3) as compared to control group. 4. Discussion There is a lack of knowledge of immunotoxic potential of prometryne subchronic exposure especially on induction of apoptosis in major organs of immune system. Some authors recorded the reduction of leukocyte number and the increased DNA damage in leukocytes after exposure to prometryne (Gzhegotskii, 1968; Kulakov, 1970; Giurgea et al., 1981). Our study was designed to simulate the normal entry route (oral) of environmentally present prometryne and repeated the dosing every 48 h to maintain the constant bioavailable concentration within the tissues as previously shown by Maynard et al. (1999). Organs considered are recommended by OECD standards and immunotoxicity testing protocols (OECD, 1995). The level of the apoptosis and necrosis was determined in a point of time (after 28 days) and significant differences were compared between controls and treated groups respectively. Flow cytometry assay with annexinV-FITC and PI stain was selected as sensitive method to record early apoptotic and necrotic events. This technique is based on the observation that dying cells that undergo the stages of apoptosis display phagocytotic molecules, such as phosphatidylserine ligand of annexinV, on cell surface (Li et al., 2003).
Animals of all groups had negligible body weight gain over 28 days even in control group (Table 1). Lack of weight gain could be explained with stress during handling procedure and use of gastric canila in case of control group. In treated animals the usually expected weight loss (Pelletier et al., 2003; Tremblay et al., 2004; Aggarwal et al., 2008) was not observed because doses did not cause severe systemic toxicity and metabolic misbalance. Selected doses did not cause relentless damage to the animals tr provoke false positive immunosupression due to malnutrition like for example in Kubosaki et al. (2008) who reported that nutritional suppression might have influenced the immunological changes that appeared after increasing concentration of xenobiotic from a certain dose. Splenocytes show no change in apoptosis level regardless of the dose and do not alter the ratio of living, apoptotic or late apoptotic cells after 28 days of exposure to prometryne. In general, it seems that spleen stayed unaffected by means of cell death (either apoptotic or necrotic) except in the highest dose of exposure to prometryne. This could be the result of the trait of organ itself which accumulate dying cells and has a high ability to remove damaged and dead cells and their components (greater clearance ratio in time). Because of this ability to resolve all sorts off dying/damaged cells, at highest dosage groups the cells prone or entering programmed death or necrosis in spleen are already dissolved or cleared before the time of measurement. Handy et al. (2002) refer to the similar pathological pattern and difference between lymph node, thymus and spleens reaction to pesticides in an organ specific manner. He argues that the oxidant damage to DNA could relate to the induction of antioxidant enzymes, where the spleen appears to be different from the thymus or lymph nodes. Overall, his data suggest that the post-exposure histochemical changes are organ-
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specific, and that spleen is not representative as the other lymphoid organs when examining immunotoxicity. Clearing ability could also be the reason of observed decrease in spleen weight predominantly pronounced in the highest dose as a possible consequence of splenocyte depletion and organ atrophy, also described by Offner et al. (2006). This is in agreement with Aggarwal et al. (2008) where none of the treatments altered the absolute body weight or body weight gain. Kim et al. (2007) showed significant decreases in absolute spleen weight, but body weight, thymic weight, splenic and thymic subsets were not affected. Results observed in our study show that prometryne affects vitality (unstained cells) in thymus and lymph node. In comparison of the two affected organs (thymus, lymph node), there is the comparable but not identical pattern in decrease of cell vitality proportional to the doses which was equivalent to findings of other authors (Handy et al., 2002). The highest dose of prometryne was the most effective in reducing the percentage of live cells and at the same time increasing the level of all types of dying cells (apoptotic, late apoptotic, early necrotic and necrotic). Furthermore, vitality of lymph node cells seem to be exceedingly affected, since the decline in percentage of vital cells is more pronounced than in thymic cells after subchronic exposure to the highest dose. The described sensibility of lymph nodes is a result of a primary contact with chemicals entering circulation after absorption and therefore exposed to higher bioavilable concentrations. Lymph nodes are the major route of entry for antigens and pathogens, via the afferent lymph flow, and they can be sensitive indicators of compounds with regional or systemic immunomodulatory/toxic effects (Tuschl et al., 2002; Elmore, 2006). Furthermore, Handy et al. (2002) report that the lymph node showed the worst pathologies of all organs examined, presumably relating to their proximity to the gut and route of xenobiotic entry. Consequently, effects measured in lymph nodes in our research are more pronounced than in other organs distant in circulation of body fluids. Lower dose was sufficient to act as a primary signal for induction of early expression of apoptotic characteristics in thymus and lymph node but still (until the 28th day) did not have the potency to provoke necrotic characteristics or severely and uncontrollably eradicate cell populations (by necrosis). Medium dose had the effectiveness, which the lower dose did not, to shift the ratio of cell populations in lymph node in favour of late apoptosis and necrosis. In thymus however, in the middle dose group, the subpopulation of cells marked as early apoptotic and late apoptotic/early necrotic is missing and only a higher percentage of necrosis was noted which is similar to the findings of Prater et al. (2002). The described difference in pattern of recorded dying subpopulations in middle dose, between the two organs suggest that the cells of thymus went through the apoptotic phase few days earlier and by the 28th day the cell membrane is seriously damaged and more permeable to PI so that the majority is grouped in the necrotic subpopulation. Giurgea et al. (1981) used two fold lower doses than our lowest dose in two fold time period of exposure to prometryne than ours and recorded the increase in DNA damage in thymus. High and rapid induction of necrosis by 375 mg kg−1 , in thymus, in this experiment could be the consequence of large number of young and hence more sensible cells to chemically induced apoptosis as other authors also reported for different pesticides (Raffray and Cohen, 1991; Tebourbu et al., 1998; Gogal et al., 2000; Lee et al., 2005). If the measurement for thymus treated with the middle dose was taken earlier than 28th day, than the ratios of the percentages of dying cells would probably look similar to distribution of percentages in lymph node on the 28th day. In lymph node however, in middle dose by the 28th day apoptotic cells are not recorded although cells are still presenting phosphatidylserine, but a membrane is seriously damaged and more permeable to PI. The previous
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applies also for the lymph node. If the measurement was taken earlier in the middle dose group, apoptotic changes would be recorded in lymph node as similar to the picture of percentage ratios of the lowest dose group. This also explains why there is a noted increase in apoptosis percentage in the lowest dose but in the middle dose the whole cell subpopulation with apoptosis is missing. Therefore, three organs treated within the same doses reacted in different time dependent pattern, characteristic for each dose respectively and should be analysed as a dynamic system over time. It is known that immune system is sensitive to chemically induced apoptosis at low dose levels when no other systemic toxicity is evident (Olgun et al., 2004). Our results also show that the early apoptosis in both affected organs is already manifested after only 28 days of exposure (relatively short time) to the lowest dose without severe increase of entry in necrosis or necrosis. An elevated extent of necrotic alterations was caused by the middle and above all by the high dose. Prometryne in subchronicaly repeated doses, caused disbalance in major organs of immune system, markedly lymph nodes and thymus, where it induced early apoptotic changes in dose/time and organ specific manner. Conflict of interest None. Acknowledgements Authors wish to thank to the Ministry of Science, Education and Sports of the R. Croatia for the grant and project support. Also we wish to show our deep appreciation to Mrs. Marija Potoˇcic´ and Mrs. Anica Remenar for technical support and to retired Prof. Oscar P. Springer for the moral support and advices. Grant sponsor: Ministry of Science, Education and Sports of the R. Croatia (Grant no. 119-00000001255). References Adams, N.H., Levi, P., Hodgeson, E., 1990. In vitro study of the metabolism of atrazine, simazine, and terbutryn in several vertebrate species. J. Agric. Food Chem. 38, 1411–1417. Aggarwal, M., Naraharisetti, S.B., Dandapat, S., Degen, G.H., Malik, J.K., 2008. Perturbations in immune responses induced by concurrent subchronic exposure to arsenic and endosulfan. Toxicology 251 (1–3), 51–60. Amador-Ramírez, M.D., Mojarro-Dávila, F., Velásquez-Valle, R., 2007. Efficacy and economics of weed control for dry chile pepper. Crop Prot. 26 (4), 677–682. Balduini, L., Matoga, M., Cavalli, E., Seilles, E., Riethmuller, D., Thomassin, M., Guillaume, Y.C., 2003. Triazinic herbicide determination by gas chromatography– mass spectrometry in breast milk. J. Chromatogr. B 794 (2), 389–395. Bardalaye, P.C., Wheeler, W.B., 1985. Capillary gas chromatographic determination of prometryn and its degradation products in parsley. J. Assoc. Anal. Chem. 68 (4), 750–753. Berg, M., Müller, S.R., Schwarzenbach, R.P., 1995. Simultaneous determination of triazines including atrazine and their major metabolites hydroxyatrazine, desethylatrazine, and deisopropylatrazine in natural waters. Analyt. Chem. 67 (11), 1860–1865. Boobis, A.R., Ossendorp, B.C., Banasiak, U., Hamey, P.Y., Sebestyen, I., Moretto, A., 2008. Cumulative risk assessment of pesticide residues in food. Toxicol Lett. 15 (180), 137–150. Brusick, J.D., 1994. An assessment of the genetic toxicity of atrazine: relevance to human health and environmental effects. Mut. Res. 317, 133–144. ´ D., Zˇ idovec-Lepej, S., Remenar, A., Horvat-Kneˇzevic, ´ A., Benkovic, ´ V., Lisiˇcic, ´ D., Ðikic, Sajli, L., Springer, O.P., 2009. The effect of prometryne on subchronically treated mice evaluated by SCGE assay. Acta Biol. Hum. 60 (1), in press. Dubenetskaia, M.M., Patent, R.L., Voitik, N.P., Voinova, I.V., Krasnaia, S.D., 1981. Nutritive value of carrots grown using the herbicide prometryne. Vopr. Pitan. 6, 50–51. Dunkelberg, H., Fuchs, J., Hengstler, J.G., Klein, E., Strueder, K., 1994. Genotoxic effects of the herbicides alachlor, atrazine, pendimethalmine, and simazine in a mammalian cells. Bull. Envirom. Contam. Toxicol. 52, 498–504. Egert, G., Greim, H., 1976. Formation of mutagenic N-nitroso compounds from the pesticides prometryne, dodine and carbaryl in the presence of nitrite at pH 1. Mutat. Res. 37 (2–3), 179–186. Elmore, S.A., 2006. Enhanced histopathology of the lymph nodes. Toxicol Pathol. 34 (5), 634–647.
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Effects of prometryne on apoptosis and necrosis in thymus, lymph node and spleen in mice Domagoj Ðikic´ a,∗ , Snjeˇzana Zˇ idovec-Lepej b , Anica Remenar b , Kreˇso Bendelja c , Vesna Benkovic´ a , Anica Horvat-Kneˇzevic´ a , Gordana Brozovic´ d , Nada Orˇsolic´ a a
Faculty of Science, University of Zagreb, Department of Animal Physiology, Biology Division, Rooseveltov trg 6, HR-10000 Zagreb, Croatia Molecular Diagnostics Laboratory of University Hospital for Infectious Diseases “Dr. Fran Mihaljevi´c”, Mirogojska 8, 10000 Zagreb, Croatia Institute of Immunology, Rockefellerova 2, 10000 Zagreb, Croatia d University Hospital for Tumors, Ilica 197, HR-10000 Zagreb, Croatia b c
a r t i c l e
i n f o
Article history: Received 15 July 2008 Received in revised form 28 September 2008 Accepted 4 October 2008 Available online 15 October 2008 Keywords: Prometryne Immunotoxicity CBA mice Triazine Flow cytometry
a b s t r a c t Prometryne is a methylthio-s-triazine herbicide. Significant traces are documented in environment, mainly waters, soil and plants used for nutrition. The aim of this study was to estimate prometryne immunotoxic properties through induction of apoptotic and/or necrotic changes in thymocytes, splenocytes and lymph node cells after repeated subchronical exposure. Three different doses of prometryne (185, 375, 555 mg kg−1 ) were applied per os every 48 h, over 28 days. Flow cytometry assay (annexinVFITC and PI) was conducted to record apoptotic and necrotic damage. In the spleen significant changes in the percentage of apoptotic cells were not detected between treated and control groups respectively. In thymus and lymph node, within the lowest dose group (185 mg kg− 1), an increase in percentage of early apoptosis without any significant increase in necrosis was detected. Medium (375 mg kg−1 ) as well as high dose triggered increase in late apoptosis in lymph node while in thymus; late apoptosis was increased only in animals exposed to the highest dose (555 mg kg−1 ). The highest applied dose, in thymus and lymph node respectively, caused a general decrease in percentage of vital cells in favour of marked increase of percentages of all types of dying cells (apoptotic, late apoptotic/early necrotic and necrotic). Prometryne caused disbalance in major organs of immune system, markedly lymph nodes and thymus, by induction of early apoptotic changes in dose/time specific manner. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The use of 1,3,5-triazine or s-triazine herbicides in agriculture has increased the levels of xenobiotic compounds in soil, water and air, and present a serious risk to the environment and human health for over three decades (Brusick, 1994). Herbicide prometryne belongs to a group of methyl-thio-s-triazines and its molecular structure is similar to renowned atrazine, differing only in sulfur instead of chlorine (Kamrin and Montgomery, 2000; LeBaron et al., 2008). Both herbicides possess similar toxicokinetic properties within mammalian organism (Adams et al., 1990; Maynard et al., 1999). Herbicide atrazine belongs to a group of chloro-striazines and different toxic effects such as genotoxicity, mutagenic, endocrine disrupting and immunotoxic properties were confirmed (Boobis et al., 2008), which finally resulted in its restricted use or complete ban in many countries (Pino et al., 1988; Dunkelberg
∗ Corresponding author. Tel.: +385 1 48 777 47/53; fax: +385 1 48 26 260. ´ E-mail address: magistar [email protected] (D. Ðikic). 1382-6689/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2008.10.002
et al., 1994; Ribas et al., 1995; Lioui et al., 1998; Gammon et al., 2005). Contrary to atrazine, only slight information on toxic effects of prometryne and other methylthio-s-triazine has been published, presumably for a belief that it is a moderately toxic pesticide related to its relatively high LD50 for mice = 3750 mg kg−1 (EPA-RED, 1996). Majority of experiments investigating toxicity of prometryne were conducted in vitro or ex vivo on experimental models such as bacteria and invertebrates where prometryne was not applied alone but rather often formulated in mixtures with other chemicals (Egert and Greim, 1976; Rozek, 1978; Kaya et al., 2000; Kurebayashi, 2005). Prometryne or 1,3,5-triazine-2,4-diamine(N,N -bis(1methylethyl)-6-(methytio) is used for the control of annual broadleaf and grass weeds in crops corn, carrots, parsley, peanuts, cotton, pigeon peas, alfa alfa, and many other cultivated plants. Prometryne and residues of prometryne can be measured in significant concentrations especially in rural areas and third world countries where its use is in considerable quantities (EPA-RED, 1996; Kamrin and Montgomery, 2000; EPA IRIS, 2001; AmadorRamírez et al., 2007; Tunku et al., 2007). It is relatively persistent in waters (Berg et al., 1995; Leh et al., 2005; Hua et al., 2006)
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and soil (Fiveland, 1977; Muller and Britz, 1982) or even in air near production or application sites (Schumann et al., 1990). Significant traces are also documented in plants used as human and domestic animal nutrition (Fiveland, 1977; Dubenetskaia et al., 1981; Bardalaye and Wheeler, 1985; Zhang et al., 2006) and some medical plants protected with herbicide prometryne (Reifenstein and Pank, 1975). Beside that traces of prometryne could be found in cow and breast milk (Krivankova et al., 1989; Balduini et al., 2003). Some investigations point to immunohematotoxicity and suppressed cellular immune response (Gzhegotskii, 1968; Kulakov, 1970; Giurgea et al., 1981). It is established that the prometryne induce DNA fragmentation, a hallmark of apoptosis in blood leukocytes (Ðikic´ et al., 2009). Therefore, this study was designed to evaluate the apoptotic and/or necrotic induction potential of prometryne in thymocytes, splenocytes and lymph node cells after repeated in vivo subchronical exposure to three different doses on mice model. Flow cytometry assay with annexinV-FITC and PI stain, as a highly sensitive method was selected to record early apoptotic and necrotic events. This technique is based on the observation that soon after initiating apoptosis, cells translocate the membrane phosphatidylserine (PS) from the inner face of the plasma membrane to the cell surface while necrotic cells become more permeable to propidium iodide. 2. Materials and methods 2.1. Animals Inbred CBA mice (65 ± 3 days old), from the mouse colony of Faculty of Science, University of Zagreb were used. The animals were maintained on a pellet diet (Pliva dd, Zagreb, Croatia) and water was provided ad libitum. The animals were maintained under 12L:12D hours light–dark regime. Experiments were carried out according to the guidelines force in Croatia (Law on the Welfare of Animals, NN # 19, 1999) and in compliance with the Guide for the Care and Use of Laboratory Animals, DHHS Publ. # (NIH) 86-123 and OECD guidelines for subchronic (28 days) toxicity testing in rodents (OECD, 1995). 2.2. Treatment of experimental animals with prometryne Mice where randomly distributed to groups of 10 individuals of both sexes. Within each group (N = 10, 5♀ + 5♂) mice were receiving low, medium and high doses of prometryne (C10 H19 N5 S), CAS No. 7287-19-6, EPA Reg. No. 9779-297 (tech. grade 95%, Herbos dd, Sisak, Croatia). Control group (N = 10, 5♀ + 5♂) received no prometryne. The prometryne doses were 185, 375 and 555 mg kg−1 respectively. The medium dose represents 1/10 of the LD50(mice) = 3750 mg kg−1 (EPA-RED, 1996). Dose of 185 mg kg−1 which is 1/20 of the LD50 and two fold lower than middle dose used, was selected as first effective dose (ED10 ) recognized through pilot tests. Dose of 555 mg kg−1 was selected as highest effective dose without lethal toxicity (LD10 ) during 28 days and also recognized through pilot tests. The metabolic study of Maynard et al. (1999) showed that the majority of the absorbed prometryne is metabolized or excreted within 48 h. To avoid overdose through bioaccumulation (log PKo/w = 3.5; Kamrin and Montgomery, 2000) mice were given prometryne per os, every 48 h, during 28 days in doses (185, 375 and 555 mg kg−1 ). Prometryne was prepared as a corn oil suspension, in volume of 0.2 ml per animal. Appropriate control group was selected and treated with the same volume of corn oil. The whole experiment was repeated three times. 2.3. Determination of body weight and organ Mice were weighed every 2 days during the experimental period starting on day 1 after application of prometryne p.o. The organs were collected on the 28th day of the experiment respectively, by protocols described in EMPReSS, SOP (Standard Operating Procedure (Green et al., 2005) but also see: http://empress.har.mrc.ac.uk) and processed to cell extractions. 2.4. Tissue cell preparation, cell staining and flow cytometry Single cell suspensions were prepared from spleen, thymus and popliteal lymph nodes of each treated animal by gently rubbing the organs through nylon mesh filter. Cellular debris was removed and cells were washed in PBS and resuspended (105 cells ml−1 ). For cell staining the standard Annexin-V-FITC Detection Kit (BioVision® ) was used according to manufacturer instructions. Briefly, 105 cells were
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resuspended in 100 L of binding buffer and 10 L of annexin-V-FITS (fluorescein isothiocyannate-labelled annexin V) and PI (propidium iodine) was added. After 15 min of incubation at 30 ◦ C, additional 400 L of binding buffer was added and reading was taken on flow cytometer FC 500 CYTOMICS Beckman Coulter, Florida, USA (EX = 488 nm, EM = 525 nm). The gate was same for both the exposed and control mice and the results are shown as percentage of positive cells within gate. Analysis was performed by CXP 2.0 software. 2.5. Statistical analysis Statistical analyses were performed using Statistica 5.0 software (StatSoft, Tulsa, USA). Each sample was characterized considering the mean (±standard deviation of the mean). The unit of measurement was the animal. Multiple comparisons between groups were done by means of ANOVA. Post hoc analysis of differences was conducted by Scheffé and Duncan test to establish the differences between groups. Level of statistical significance was set at p ≤ 0.05 and in Tables differences are marked according to LSD system.
3. Results The body weights of all experimental animals were not significantly changed during experiment (Table 1). Weight of spleen decreased significantly (p ≤ 0.05) in the group of animals receiving the highest dose (555 mg kg−1 ) while weight of thymus and lymph nodes were not significantly changed (Table 1). The percentage of unstained cells (annexinV−/PI−) was significantly decreased (p ≤ 0.05) in thymus (Table 2) and lymph node (Table 4) in the highest dose (555 mg kg−1 ) compared to the control. There was no significant difference in percentage of unstained cells in thymus (Table 2) and lymph node (Table 4) within the low (185 mg kg−1 ) and middle (375 mg kg−1 ) dose compared to the control. The percentage of unstained cells in spleen was unaffected by prometryne in any applied dose compared to control (Table 3). The percentage of cells in early apoptosis (annexinV+/PI−) was significantly increased (p ≤ 0.05) in thymus (Table 2) and lymph node (Table 4) treated with doses of 185 and 555 mg kg−1 compared to the control. while no differences were found in percentage of cells in spleen (Table 3). The percentage of cells in late apoptosis (annexinV+/PI+) was significantly increased (p ≤ 0.05) in thymus exposed to the highest dose (Table 2) compared to control animals. In lymph node significantly increased percentage of cells in late apoptosis was observed in medium and high dose groups compared to the control groups (Table 4). There were no significant changes in annexinV+/PI+ splenocytes in any of the dose groups compared to the control (Table 3).
Table 1 Body weight of mice throughout the experiment and organ in control group and groups of animals treated with three different doses of prometryne. Body weight1 (mean ± S.D.) 0 mg kg−1 Dose of prometryne 1st day (g) 21.55 ± 2.57 14th day (g) 21.57 ± 2.83 28th day (g) 21.65 ± 3.05
185 mg kg−1 21.83 ± 3.11 22.08 ± 2.88 22.40 ± 2.88
375 mg kg−1 21.47 ± 3.56 21.62 ± 3.16 21.40 ± 3.11
555 mg kg−1 21.57 ± 2.21 20.86 ± 2.17 21.83 ± 3.11
Organ weight1 (mean ± S.D.)
Dose of prometryne Thymus (mg) Spleen (mg) Lymph node (mg)
0 mg kg−1
185 mg kg−1
29.48 ± 3.91 86.24 ± 9.61b 10.25 ± 4.96
27.00 ± 7.90 25.92 ± 7.50 85.3 ± 10.2b 89.6 ± 10.0b 8.26 ± 3.49 8.30 ± 2.50
375 mg kg−1
555 mg kg−1 29.31 ± 3.34 75.7 ± 14.2a 11.16 ± 4.29
1 Body weight was measured every second day of the experiment, for simplicity only three measurements are shown. a,b Within row, means with different superscripts (letters) are significantly different (p ≤ 0.05).
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Table 2 Percentages of cells in thymus of control group and groups of animals treated with three different doses of prometryne. 0 mg kg−1
Doses of prometryne Unstained cells (annexinVFITC−/PI−) Early apoptosis (annexinVFITC+/PI−) Late apoptosis (annexinVFITC+/PI+) Necrosis (annexinVFITC−/PI+) a,b,c
185 mg kg−1
375 mg kg−1
555 mg kg−1
Mean ± S.D.
Min.
Max.
Mean ± S.D.
Min.
Max.
Mean ± S.D.
Min.
Max.
Mean ± S.D.
Min.
Max.
± ± ± ±
76.7 0.0 0.1 0.7
96.5 11.6 10.5 3.3
79.3 11.8 6.8 1.9
± ± ± ±
62.6 5.3 3.6 0.6
86.1 22.3 17.5 4.5
83.7 8.3 5.1 2.7
± ± ± ±
78.7 0.5 0.0 1.1
95.9 11.8 8.6 3.8
70.3 16.1 10.7 2.6
± ± ± ±
54.2 10.9 9.1 0.7
77.3 25.5 13.9 6.2
85.5 6.7 5.9 1.7
6.9b 4.1a 3.6a 0.9a
7.1b 5.4b 3.3a 1.0a
5.1b 3.5a 2.3a 0.9b
9.3a 5.9c 1.8b 2.1b
Within row, means with different superscripts (letters) are significantly different (p ≤ 0.05).
Table 3 Percentages of spleen cells of control group and groups of animals treated with three different doses of prometryne. 0 mg kg−1
Doses of prometryne Unstained cells (annexinVFITC−/PI−) Early apoptosis (annexinVFITC+/PI−) Late apoptosis (annexinVFITC+/PI+) Necrosis (annexinVFITC−/PI+) a,b
185 mg kg−1
375 mg kg−1
555 mg kg−1
Mean ± S.D.
Min.
Max.
Mean ± S.D.
Min.
Max.
Mean ± S.D.
Min.
Max.
Mean ± S.D.
Min.
Max.
± ± ± ±
60.0 1.7 2.4 2.8
86.7 13.4 14.8 7.0
78.1 6.6 9.0 6.0
± ± ± ±
69.6 3.3 4.6 3.0
82.1 10.6 15.9 10.3
79.1 5.0 9.6 5.7
± ± ± ±
77.5 2.2 7.8 5.1
81.9 7.4 11.1 6.4
76.6 8.8 10.7 3.7
± ± ± ±
71.9 6.0 6.1 2.7
85.0 10.9 15.4 5.0
77.1 6.6 8.7 5.1
7.9 2.8 3.7 2.2a
3.3 2.1 2.7 2.2a
1.7 1.8 1.2 0.5a
5.4 2.0 3.6 0.9b
Within row, means with different superscripts (letters) are significantly different (p ≤ 0.05).
Table 4 Percentages of lymph node cells of control group and groups of animals treated with three different doses of prometryne. 0 mg kg−1
Doses of prometryne Unstained cells (annexinVFITC−/PI−) Early apoptosis (annexinVFITC+/PI−) Late apoptosis (annexinVFITC+/PI+) Necrosis (annexinVFITC−/PI+) a,b,c
185 mg kg−1
375 mg kg−1
555 mg kg−1
Mean ± S.D.
Min.
Max.
Mean ± S.D.
Min.
Max.
Mean ± S.D.
Min.
Max.
Mean ± S.D.
Min.
Max.
± ± ± ±
77.4 1.4 1.0 0.3
94.6 14.7 19.4 1.6
83.2 10.7 5.3 0.7
± ± ± ±
64.9 5.9 2.2 0.3
88.4 15.6 6.4 1.5
77.7 7.3 13.6 1.2
± ± ± ±
71.0 6.8 9.1 0.7
82.2 7.9 20.2 1.6
69.4 13.5 15.3 1.6
± ± ± ±
58.1 12.1 9.0 0.5
75.7 14.8 25.3 2.6
83.0 8.8 7.3 0.8
7.3b 3.7a 4.6a 0.2a
3.3b 3.2b 1.2a 0.3a
5.9b 0.5a 5.8b 0.5b
7.2a 1.2b 6.3b 0.8b
Within row, means with different superscripts (letters) are significantly different (p ≤ 0.05).
The percentage of necrotic cells (annexinV−/PI+) in thymus (Table 2) and lymph node (Table 4) was significantly increased (p ≤ 0.05) in middle and high doses, while significant decrease (p ≤ 0.05) was observed in spleen (Table 3) as compared to control group. 4. Discussion There is a lack of knowledge of immunotoxic potential of prometryne subchronic exposure especially on induction of apoptosis in major organs of immune system. Some authors recorded the reduction of leukocyte number and the increased DNA damage in leukocytes after exposure to prometryne (Gzhegotskii, 1968; Kulakov, 1970; Giurgea et al., 1981). Our study was designed to simulate the normal entry route (oral) of environmentally present prometryne and repeated the dosing every 48 h to maintain the constant bioavailable concentration within the tissues as previously shown by Maynard et al. (1999). Organs considered are recommended by OECD standards and immunotoxicity testing protocols (OECD, 1995). The level of the apoptosis and necrosis was determined in a point of time (after 28 days) and significant differences were compared between controls and treated groups respectively. Flow cytometry assay with annexinV-FITC and PI stain was selected as sensitive method to record early apoptotic and necrotic events. This technique is based on the observation that dying cells that undergo the stages of apoptosis display phagocytotic molecules, such as phosphatidylserine ligand of annexinV, on cell surface (Li et al., 2003).
Animals of all groups had negligible body weight gain over 28 days even in control group (Table 1). Lack of weight gain could be explained with stress during handling procedure and use of gastric canila in case of control group. In treated animals the usually expected weight loss (Pelletier et al., 2003; Tremblay et al., 2004; Aggarwal et al., 2008) was not observed because doses did not cause severe systemic toxicity and metabolic misbalance. Selected doses did not cause relentless damage to the animals tr provoke false positive immunosupression due to malnutrition like for example in Kubosaki et al. (2008) who reported that nutritional suppression might have influenced the immunological changes that appeared after increasing concentration of xenobiotic from a certain dose. Splenocytes show no change in apoptosis level regardless of the dose and do not alter the ratio of living, apoptotic or late apoptotic cells after 28 days of exposure to prometryne. In general, it seems that spleen stayed unaffected by means of cell death (either apoptotic or necrotic) except in the highest dose of exposure to prometryne. This could be the result of the trait of organ itself which accumulate dying cells and has a high ability to remove damaged and dead cells and their components (greater clearance ratio in time). Because of this ability to resolve all sorts off dying/damaged cells, at highest dosage groups the cells prone or entering programmed death or necrosis in spleen are already dissolved or cleared before the time of measurement. Handy et al. (2002) refer to the similar pathological pattern and difference between lymph node, thymus and spleens reaction to pesticides in an organ specific manner. He argues that the oxidant damage to DNA could relate to the induction of antioxidant enzymes, where the spleen appears to be different from the thymus or lymph nodes. Overall, his data suggest that the post-exposure histochemical changes are organ-
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specific, and that spleen is not representative as the other lymphoid organs when examining immunotoxicity. Clearing ability could also be the reason of observed decrease in spleen weight predominantly pronounced in the highest dose as a possible consequence of splenocyte depletion and organ atrophy, also described by Offner et al. (2006). This is in agreement with Aggarwal et al. (2008) where none of the treatments altered the absolute body weight or body weight gain. Kim et al. (2007) showed significant decreases in absolute spleen weight, but body weight, thymic weight, splenic and thymic subsets were not affected. Results observed in our study show that prometryne affects vitality (unstained cells) in thymus and lymph node. In comparison of the two affected organs (thymus, lymph node), there is the comparable but not identical pattern in decrease of cell vitality proportional to the doses which was equivalent to findings of other authors (Handy et al., 2002). The highest dose of prometryne was the most effective in reducing the percentage of live cells and at the same time increasing the level of all types of dying cells (apoptotic, late apoptotic, early necrotic and necrotic). Furthermore, vitality of lymph node cells seem to be exceedingly affected, since the decline in percentage of vital cells is more pronounced than in thymic cells after subchronic exposure to the highest dose. The described sensibility of lymph nodes is a result of a primary contact with chemicals entering circulation after absorption and therefore exposed to higher bioavilable concentrations. Lymph nodes are the major route of entry for antigens and pathogens, via the afferent lymph flow, and they can be sensitive indicators of compounds with regional or systemic immunomodulatory/toxic effects (Tuschl et al., 2002; Elmore, 2006). Furthermore, Handy et al. (2002) report that the lymph node showed the worst pathologies of all organs examined, presumably relating to their proximity to the gut and route of xenobiotic entry. Consequently, effects measured in lymph nodes in our research are more pronounced than in other organs distant in circulation of body fluids. Lower dose was sufficient to act as a primary signal for induction of early expression of apoptotic characteristics in thymus and lymph node but still (until the 28th day) did not have the potency to provoke necrotic characteristics or severely and uncontrollably eradicate cell populations (by necrosis). Medium dose had the effectiveness, which the lower dose did not, to shift the ratio of cell populations in lymph node in favour of late apoptosis and necrosis. In thymus however, in the middle dose group, the subpopulation of cells marked as early apoptotic and late apoptotic/early necrotic is missing and only a higher percentage of necrosis was noted which is similar to the findings of Prater et al. (2002). The described difference in pattern of recorded dying subpopulations in middle dose, between the two organs suggest that the cells of thymus went through the apoptotic phase few days earlier and by the 28th day the cell membrane is seriously damaged and more permeable to PI so that the majority is grouped in the necrotic subpopulation. Giurgea et al. (1981) used two fold lower doses than our lowest dose in two fold time period of exposure to prometryne than ours and recorded the increase in DNA damage in thymus. High and rapid induction of necrosis by 375 mg kg−1 , in thymus, in this experiment could be the consequence of large number of young and hence more sensible cells to chemically induced apoptosis as other authors also reported for different pesticides (Raffray and Cohen, 1991; Tebourbu et al., 1998; Gogal et al., 2000; Lee et al., 2005). If the measurement for thymus treated with the middle dose was taken earlier than 28th day, than the ratios of the percentages of dying cells would probably look similar to distribution of percentages in lymph node on the 28th day. In lymph node however, in middle dose by the 28th day apoptotic cells are not recorded although cells are still presenting phosphatidylserine, but a membrane is seriously damaged and more permeable to PI. The previous
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applies also for the lymph node. If the measurement was taken earlier in the middle dose group, apoptotic changes would be recorded in lymph node as similar to the picture of percentage ratios of the lowest dose group. This also explains why there is a noted increase in apoptosis percentage in the lowest dose but in the middle dose the whole cell subpopulation with apoptosis is missing. Therefore, three organs treated within the same doses reacted in different time dependent pattern, characteristic for each dose respectively and should be analysed as a dynamic system over time. It is known that immune system is sensitive to chemically induced apoptosis at low dose levels when no other systemic toxicity is evident (Olgun et al., 2004). Our results also show that the early apoptosis in both affected organs is already manifested after only 28 days of exposure (relatively short time) to the lowest dose without severe increase of entry in necrosis or necrosis. An elevated extent of necrotic alterations was caused by the middle and above all by the high dose. Prometryne in subchronicaly repeated doses, caused disbalance in major organs of immune system, markedly lymph nodes and thymus, where it induced early apoptotic changes in dose/time and organ specific manner. Conflict of interest None. Acknowledgements Authors wish to thank to the Ministry of Science, Education and Sports of the R. Croatia for the grant and project support. Also we wish to show our deep appreciation to Mrs. Marija Potoˇcic´ and Mrs. Anica Remenar for technical support and to retired Prof. Oscar P. Springer for the moral support and advices. Grant sponsor: Ministry of Science, Education and Sports of the R. Croatia (Grant no. 119-00000001255). References Adams, N.H., Levi, P., Hodgeson, E., 1990. In vitro study of the metabolism of atrazine, simazine, and terbutryn in several vertebrate species. J. Agric. Food Chem. 38, 1411–1417. Aggarwal, M., Naraharisetti, S.B., Dandapat, S., Degen, G.H., Malik, J.K., 2008. Perturbations in immune responses induced by concurrent subchronic exposure to arsenic and endosulfan. Toxicology 251 (1–3), 51–60. Amador-Ramírez, M.D., Mojarro-Dávila, F., Velásquez-Valle, R., 2007. Efficacy and economics of weed control for dry chile pepper. Crop Prot. 26 (4), 677–682. Balduini, L., Matoga, M., Cavalli, E., Seilles, E., Riethmuller, D., Thomassin, M., Guillaume, Y.C., 2003. Triazinic herbicide determination by gas chromatography– mass spectrometry in breast milk. J. Chromatogr. B 794 (2), 389–395. Bardalaye, P.C., Wheeler, W.B., 1985. Capillary gas chromatographic determination of prometryn and its degradation products in parsley. J. Assoc. Anal. Chem. 68 (4), 750–753. Berg, M., Müller, S.R., Schwarzenbach, R.P., 1995. Simultaneous determination of triazines including atrazine and their major metabolites hydroxyatrazine, desethylatrazine, and deisopropylatrazine in natural waters. Analyt. Chem. 67 (11), 1860–1865. Boobis, A.R., Ossendorp, B.C., Banasiak, U., Hamey, P.Y., Sebestyen, I., Moretto, A., 2008. Cumulative risk assessment of pesticide residues in food. Toxicol Lett. 15 (180), 137–150. Brusick, J.D., 1994. An assessment of the genetic toxicity of atrazine: relevance to human health and environmental effects. Mut. Res. 317, 133–144. ´ D., Zˇ idovec-Lepej, S., Remenar, A., Horvat-Kneˇzevic, ´ A., Benkovic, ´ V., Lisiˇcic, ´ D., Ðikic, Sajli, L., Springer, O.P., 2009. The effect of prometryne on subchronically treated mice evaluated by SCGE assay. Acta Biol. Hum. 60 (1), in press. Dubenetskaia, M.M., Patent, R.L., Voitik, N.P., Voinova, I.V., Krasnaia, S.D., 1981. Nutritive value of carrots grown using the herbicide prometryne. Vopr. Pitan. 6, 50–51. Dunkelberg, H., Fuchs, J., Hengstler, J.G., Klein, E., Strueder, K., 1994. Genotoxic effects of the herbicides alachlor, atrazine, pendimethalmine, and simazine in a mammalian cells. Bull. Envirom. Contam. Toxicol. 52, 498–504. Egert, G., Greim, H., 1976. Formation of mutagenic N-nitroso compounds from the pesticides prometryne, dodine and carbaryl in the presence of nitrite at pH 1. Mutat. Res. 37 (2–3), 179–186. Elmore, S.A., 2006. Enhanced histopathology of the lymph nodes. Toxicol Pathol. 34 (5), 634–647.
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