- Email: [email protected]
Effects of organophosphorus pesticides and their ozonation byproducts on gap junctional intercellular communication in rat liver cell line
Food and Chemical Toxicology 45 (2007) 2057–2063 www.elsevier.com/locate/foodchemtox
Effects of organophosphorus pesticides and their ozonation byproducts on gap junctional intercellular communication in rat liver cell line Jiguo Wu a
a,c
, Li Lin a, Tiangang Luan
a,*
, Yuk Sing Chan Gilbert b, Chongyu Lan
a,*
State Key Laboratory of Biocontrol, School of Life Sciences, Zhongshan (Sun Yat-sen) University, Guangzhou 510275, PR China b Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hong Kong, PR China c School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, PR China Received 15 June 2006; accepted 10 May 2007
Abstract The effects of organophosphorus pesticides (OPs), oxons and their ozonation byproducts on gap junctional intercellular communication (GJIC) on cultured BRL cell line were investigated using scrape loading and dye transfer (SL/DT) technique. The neutral red uptake assay was used to identify the non-cytotoxic levels of diazinon, parathion and methyl-parathion applied to GJIC assay. The concentration-dependent inhibition of GJIC was observed over a range of 50–350 mg/l diazinon, parathion and methyl-parathion after 90 min incubation compared with the vehicle control. However, oxons and ozonation byproducts of OPs had no inhibition effect on GJIC at any of the concentrations tested. The inhibition of GJIC by OPs was reversible after removal of the tested pesticides followed by incubation with fresh medium. The present study suggested that the ozonation treatment could be used for the detoxification of drinking water and food crops contaminated with diazinon, parathion and methyl-parathion without formation of GJIC toxicity. 2007 Elsevier Ltd. All rights reserved. Keywords: Diazinon; Parathion; Methyl-parathion; Ozone; Buffalo rat liver
1. Introduction Organophosphorus pesticides (OPs) are one of the most important groups of insecticides and are widely applied to pest control in agriculture and hygiene. The extensive application of OPs can subsequently release OPs into hydrological systems (Thurman and Meyer, 1996; Galindo-Reyes et al., 1999; Tariq et al., 2004) or contaminate food crops (Hura et al., 1999; Gupta, 2004; Bai et al., 2006), which may pose a seriously adverse impact to non-
Abbreviations: Buffalo rat liver (BRL); Dulbecco’s Modified Eagle Medium (DMEM); Gap junctional intercellular communication (GJIC); Organophosphorus pesticide (OP); Phosphate buffered saline (PBS); Scrape loading and dye transfer (SL/DT). * Corresponding authors. Tel.: +86 20 84036296; fax: +86 20 84113652. E-mail address: [email protected] (T. Luan). 0278-6915/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2007.05.006
target organisms and humans due to various toxicities of OPs. OPs elicit their extensive toxicities through various mechanisms and pathways. Primarily, OPs elicit their acute toxicities through a common mechanism of inhibition of acetylcholinesterase, ultimately modifying cholinergic signaling through disruption of acetylcholine degradation (Pope et al., 2005). OPs also elicit their toxicities via other mechanisms including cytotoxicity (Carlson and Ehrich, 1999; Wagner et al., 2005), genotoxicity (Błasiak et al., 1999; Das and John, 1999; Rahman et al., 2002), disruption of sex hormone and reproductivity (Kang et al., 2004; Okamura et al., 2005), and immunotoxicity (Navarro et al., 2001; Yeh et al., 2005). Gap junctional intercellular communication (GJIC) has been considered as an important determinant for normal cell growth, development, differentiation as well as
2058
J. Wu et al. / Food and Chemical Toxicology 45 (2007) 2057–2063
maintenance of homeostasis in multicellular organisms. Inhibition of GJIC by various chemicals has been postulated to be a factor in the tumor promotion phase of carcinogenesis, teratogenesis, neurotoxicity, reproductive dysfunction and other chemically-induced disease states (El-Fouly et al., 1987). Untimely or chronic disruption of GJIC during embryonic or fetal development could lead to embryonic lethality or teratogenesis and chemical modulation of GJIC in initiated tissues has been shown to lead to tumor promotion (Trosko et al., 1998). GJIC assay has been shown to be a reliable, simple, inexpensive in vitro assay as well as a good predictor of epigenetic toxicants (Rosenkranz et al., 2000), therefore it has been a useful marker for testing the epigenetic toxicity of some toxicants such as polycyclic aromatic hydrocarbons (PAHs), organochlorine compounds (Upham et al., 1996, 1998; Tiemann and Po¨hland, 1999; Tiemann et al., 2002) and the ozonation byproducts or biometabolites of the parent compounds (Upham et al., 1995; Ghoshal et al., 1999; Herner et al., 2001; Masten et al., 2001; Luster-Teasley et al., 2002). Ozone was widely used in drinking water treatment for disinfection and oxidation of contaminant. Ozonation treatment can be used to oxidize OPs in aqueous solution (Chiron et al., 1998; Ku et al., 1998; Masten et al., 2001; Benitez et al., 2002; Ku and Lin, 2002). Ozone has also been applied to remove residual pesticides in food crops (Ong et al., 1996; Hwang et al., 2001; Wu et al., 2007) as well as food hygiene and sanitation (Guzel-Seydim et al., 2004). However, the target compounds will not be mineralized, but transformed to metabolites of lesser molecular weight in actual ozone application (Gottschalk et al., 2000). So toxicity assessments of both parent OPs and their ozonation byproducts are necessary for the sake of safety consideration after ozonation treatment. The objective of the present study was (1) to investigate the effects of OPs and their ozonation byproducts on GJIC; and (2) to evaluate the safety of ozonation treatment of OPs using GJIC bioassay. 2. Methods and materials 2.1. Chemicals and reagents The pesticide standards of diazinon (CAS#: 333-41-5; purity: 98.1%), parathion (CAS#: 56-38-2; purity: 98%), paraoxon (CAS#: 311-45-5; purity: 98.6%), methyl-parathion (CAS#: 298-00-0; purity: 99.9%) and methyl-paraoxon (CAS#: 950-35-6; purity: 99.0%) were from Supelco, USA. Diazinon oxon (CAS#: 962-58-3; purity: 99.0%) was from Wako, Japan. All pesticide standard stock solutions (20 mg/ml) were prepared in acetone. Analytical grade acetone and ethyl acetate (CAS#: 141-78-6) (Guangzhou Chemical Factory, China) were redistilled before use. Neutral red was from Tianjing Chemical Development Center, China. Trypsin solution (0.25%) was from GIBCO, USA. Lucifer yellow CH was from Invitrogen, USA, and dissolved in phosphate buffered saline (PBS) (pH 7.2) with concentration of 1 mg/ml.
2.2. Preparation of the ozonation byproducts of OPs Ozonation experiments were carried out by bubbling gaseous ozone of 5.5 mg/l at a flow rate of 90 ml/min into a 250 ml glass column through a
disperser at the column bottom for preparation of ozonation byproducts of the target OPs. Two hundred ml of individual OP solution (initial concentration: 200 lg/ml) was ozonated for various time periods. The excess gaseous ozone was trapped in 2% potassium iodide (KI) solution. After ozonation of diazinon and parathion solution for 180 min and methyl-parathion solution for 210 min (more than 99% of parent pesticides was degraded (data not shown)), 100 ml samples were collected and 99.999% purity nitrogen was bubbled into the samples for 2 min to quench the reactions between ozone and pesticides. The samples were transferred to 250 ml separated funnels and extracted three times with 100 ml ethyl acetate. The combined extracts were evaporated to approximately 5 ml by the rotary evaporator (BUCHI, Switzerland) followed by purging of the remaining ethyl acetate with high purity nitrogen, then the residual was reconstituted in 0.5 ml acetone. The control sample was prepared with ultra-pure water (Millipore system, USA) without addition of pesticide according to the same procedure. The parent pesticide concentration was used as the basis to calculate the concentration of ozonation byproducts.
2.3. Cell culture The buffalo rat liver (BRL) cell line from Experimental Animal Center of Sun Yat-sen University were grown in Dulbecco’s Modified Eagle Medium (DMEM) (GIBCO, USA), supplemented with 5% newborn calf serum (Sijiqing, Hangzhou, China), 100 U/ml penicillin and streptomycin 100 lg/ml and incubated at 37 C in a humidified atmosphere containing 5% (v/v) CO2 and 95% air. The cells were grown in 25 mm2 tissue culture flasks or tissue culture dishes of 40 · 10 mm (TPP, Europe/Switzerland) or 96-well plates (TPP, Europe/Switzerland). The cell cultures were subcultured in fresh DMEM every three days.
2.4. Neutral red uptake assay The cytotoxicities of the OPs and their ozonation byproducts were tested by neutral red uptake assay developed by Borenfreund and Puerner (1985) and modified by Masten et al. (2001). The procedure is briefly described as follows: Neutral red dye stock solution (0.4%) was prepared in PBS and filtered with 0.22 lm syringe-filter (TPP, Europe/Switzerland). This neutral red solution was diluted to a final working concentration of 0.04% in DMEM. The diluted solution was incubated at 37 C overnight followed by filtration with 0.22 lm syringe-filter to remove fine precipitates. Cells were cultured in 96-well plates for two days. After incubation with DMEM containing various concentrations of OPs or the ozonation byproducts or vehicle control for 3 h, the cells were rinsed three times with PBS and incubated with DMEM containing 0.04% neutral red dye for 3 h. Then the medium was discarded and the cells were rinsed three times with PBS. Subsequently, 0.2 ml solution containing 1% acetic acid and 50% ethanol was added to each well of the 96-well plates to extract neutral red dye for 20 min. Wellscan MK3 (Labsystems Dragon, Finland) was used to determine the absorbance of neutral red dye released from the cells in each well of the 96-well plates at a wavelength of 550 nm. Every treatment was repeated four times. The cytotoxicities of the target pesticides and their ozonation byproducts were reported as relative viability to the control.
2.5. GJIC assay GJIC assay was carried out in 40 · 10 mm tissue culture dishes with 100% confluent monolayer cells grown in two ml DMEM supplemented with 5% newborn calf serum, 100 U/ml penicillin and streptomycin 100 lg/ml. GJIC was detected using the scrape loading and dye transfer (SL/DT) technique developed by El-Fouly et al. (1987). Assays for different treatments and vehicle control were run in triplicate tissue culture dishes. Monolayer cells with 100% confluence were incubated with target compounds for a constant time period with different concentrations or for various time periods with constant concentration. After exposure to the target compounds, the cells were rinsed three times with PBS and one ml of lucifer yellow CH solution was added to the cell cultures and scrape-
J. Wu et al. / Food and Chemical Toxicology 45 (2007) 2057–2063 Table 1 The evaluation criterion of inhibitory GJIC
a
Relative migration to the control (%)
Response
Reference
<30
Complete inhibition Partial inhibition No inhibition
Herner et al. (2001)
30–90 >90
2059
120 100 80 60
Diazinon Methyl parathion Parathion
40 20 0
b
120
Relative viability
0 loaded with several scrapes using a steel surgical blade (El-Fouly et al., 1987). The dye solution was left on the cell cultures for 3 min, then discarded and the cell cultures were carefully rinsed three times with PBS to remove detached cells and background fluorescence. Several drops of 4% formalin in PBS were added to fix the cell cultures. An inverted fluorescence microscope equipped with a digital camera (Nikon Eclipse TE 2000U system, Nikon ACT-1 version 2.62, Nikon corporation, Japan) was used to record the migration of the lucifer yellow CH dye from the edge cells of the scrape. The migration was measured on the micrograph. An average value of 30 measurements for each treatment (10 measurements per dish) was regarded as the migration of the dye in the cell cultures. The percentage of the migration of the dye in the cell cultures exposed to target compounds to the migration which the dye traveled in the vehicle control was used to evaluate the inhibition of GJIC. Table 1 shows the evaluation criterion of inhibitory GJIC proposed by Herner et al. (2001).
100
50
The cells were cultured under the same conditions as the GJIC assay and incubated in DMEM containing the target pesticides or ozonation byproducts with constant concentration for 90 min. After removing the target compounds, the cells were rinsed three times with PBS and incubated in fresh DMEM for 30, 60, 90, 120, 150 min, respectively. These cell cultures were then used to determine GJIC function according to the SL/ DT technique described in GJIC assay. Every treatment was conducted in triplicate.
150
200
250
300
350
400
200
250
300
350
400
300
350
400
80 60
Diazinon oxon Methyl paraoxon Methyl paraoxon
40 20 0
c
120
0
50
100
150
100 80 60 Diazinon byproducts Methyl parathion byproducts Parathion byproducts
40
2.6. GJIC recovery test
100
20 0 0
50
100
150
200
250
(mg/ml) Fig. 1. Concentration effects on cytotoxicity. The cell cultures were exposed to (a) parent pesticides diazinon, methyl-parathion and methylparathion; (b) oxons (diazinon oxon, methyl-paraoxon and paraoxon); (c) ozonation byproducts of diazinon, methyl-parathion and parathion for 3 h. The error bars were one standard deviation (n = 4).
2.7. Data analysis The results were reported as a mean ±1 SD. A t-test was performed to determine whether different treatments differed significantly from the control at p 6 0.05, using the software SAS (SAS Institute Inc., Cary, NC, USA.).
3. Results 3.1. Cytotoxicity assay In order to identify the non-cytotoxic levels of the selected OPs applied to GJIC studies, neutral red uptake assay was used to determine the cytotoxicity of the target compounds. Diazinon, parathion and methyl-parathion were non-cytotoxic at the tested levels (10–350 mg/l) after incubation for 3 h (Fig. 1a). And diazinon oxon, paraoxon and methyl-paraoxon were also non-cytotoxic at concentrations of 50–400 mg/l (Fig. 1b). Fig. 1c also indicates that the ozonation byproducts were non-cytotoxic for all of the concentrations tested (50–400 mg/l). 3.2. Concentration effect on GJIC For evaluation of concentration response, GJIC levels were detected after the cells were exposed to the target
compounds with non-cytotoxic levels for 90 min. Methylparathion inhibited GJIC over a concentration range of 150–300 mg/l (Fig. 2a) and partially inhibited GJIC was found (84.4% to the control) at the concentration of 150 mg/l. Almost completely inhibited GJIC was found at 300 mg/l (32.4% to the control). The results indicated that GJIC was inhibited by methyl-parathion in a concentration dependent manner (p < 0.05), beginning at 150 mg/ l. However, methyl-paraoxon and ozonation byproducts of methyl-parathion did not inhibit GJIC at any of the concentrations tested (p > 0.05). Parathion concentration-dependent (p < 0.05) inhibited GJIC over a concentration range of 100–350 mg/l (Fig. 2b). GJIC was partially inhibited at a concentration of 100 mg/l (88.8% to the control); and almost completely inhibited at concentration of 350 mg/l (32% to the control). However, paraoxon and ozonation byproducts of parathion did not inhibit GJIC at any of the concentrations tested (p > 0.05). Diazinon inhibited GJIC over a concentration range of 50–300 mg/l, while diazinon oxon and ozonation byproducts of diazinon did not inhibit GJIC at any of the concentrations tested (Fig. 2c). GJIC was also inhibited by diazinon at a concentration dependent manner (p < 0.05).
2060
120
120 100 80 60 40 20 0
100
Methyl parathion Methyl paraoxon Methyl parathion byproducts
0
50
100
150
200
250
300
350
400
Relative GJIC inhibition
b 120 100 80 60 40 20 0
c
120
Relative GJIC inhibition
a
J. Wu et al. / Food and Chemical Toxicology 45 (2007) 2057–2063
80 60 40 Diazinon Methyl parathion Parathion
20 Parathion Paraoxon Parathion byproducts
0
50
100
150
0 0 200
250
300
350
400
100 80
20
40
60
80 100 120 140 Time (min)
160 180
Fig. 3. Time effects of OPs on GJIC. The cell cultures were incubated for various time periods with DMEM containing 250 mg/l diazinon, methylparathion and parathion. The error bars were one standard deviation (n = 30).
60 40
110
Diazinon Diazinon oxon Diazinon byproducts
0 0
50
100
150
Diazinon Methyl parathion Parathion
100 200
250
300
350
400
(mg/ml) Fig. 2. Concentration effects on GJIC. Cell cultures were exposed to (a) methyl-parathion, methyl-paraoxon and ozonation byproducts of methylparathion; (b) parathion, paraoxon and ozonation byproducts of parathon; (c) diazinon, diazinon oxon and ozonation byproducts of diazinon for 90 min. The error bars were one standard deviation (n = 30).
Relative GJIC inhibition
20
90 80 70 60 50 40 30
Diazinon partially inhibited GJIC at a concentration of 50 mg/l (89.4% to the control) and almost completely inhibited GJIC at a concentration of 300 mg/l (30.4% to the control). These results suggested that the tested pesticides methylparathion, parathion and diazinon inhibited GJIC in a concentration-dependent manner, while their ozonation byproducts did not inhibit GJIC in BRL cell line.
20 -20
0
20
40
60 80 100 Time (min)
120
140
160
Fig. 4. Time of recoveries for OPs inhibitory to GJIC. The cell cultures were preincubated with DMEM containing 300 mg/l diazinon, methylparathion and 350 mg/l parathion for 90 min. The error bars were one standard deviations (n = 30).
3.3. Time effect on GJIC
methyl-parathion and parathion were recovered to 91.3%, 84.7% and 92.7%, respectively.
During all treatment times, the inhibition of GJIC was the greatest at 90 min for methyl-parathion, parathion and diazinon (Fig. 3).
4. Discussion
3.4. Recovery of GJIC The inhibition of GJIC by the tested pesticides could be time-dependent (p < 0.05) recovered after removal of the toxicants (Fig. 4). When the cells pretreated with target toxicants were incubated in the fresh medium for 150 min, the function of GJIC inhibited by diazinon,
To evaluate the epigenetic toxicity of OPs and the relevant ozonation byproducts, individual OP was ozonated and the toxicity of the mixture of byproducts produced was determined using GJIC assay in BRL cell line. As a result of ozonation of diazinon and parathion solution for 180 min and methyl-parathion solution for 210 min, more than 99% of the parent pesticides was degraded (data not shown). Generally speaking, the ozone level of 1–3 mg/ l was applied for 5–15 min in a typical full scale water treat-
J. Wu et al. / Food and Chemical Toxicology 45 (2007) 2057–2063
ment (Chu et al., 2002; Xu and Zhao, 2003), depending on the properties of water source. In nearly all cases, the target compounds will not be mineralized by ozone, but transformed to intermediates, and some of the products formed do not react further with ozone (Gottschalk et al., 2000). Therefore, the ozonation byproduct components will not be largely changed for the prolonged contact time and high initial concentration of the parent pesticide. The ozonation byproducts were extracted with ethyl acetate and reconstituted in vehicle solvent acetone which was less toxic than dimethyl sulfoxide (DMSO), the most widely used solvent in bioassay systems (Forman et al., 1999). The concentrations of the ozonation byproducts were calculated according to those of the parent pesticides. Since ozonation of organophosphorus pesticides containing P@S bond can form trace oxons as primary byproducts (Wu et al., 2007), oxons were singled out to test their toxicity. GJIC in the BRL cell line was inhibited by the selected OPs in a concentration dependent manner (p < 0.05) over the range of 50–350 mg/l. The neutral red uptake assay supported that the inhibition effect of OPs on GJIC in the BRL cell line did not result from the death of the cells. The pesticide levels at which the GJIC was most completely inhibited were 300 mg/l for methyl-parathion and diazinon (Fig. 2a and c) and 350 mg/l for parathion ( Fig. 2b). These levels were similar to that of malathion (1000 lM, equal to 330.36 mg/l), at which GJIC in BW-f343 cell line was completely inhibited (Masten et al., 2001). However, these levels of the tested pesticides were higher than that of organochlorine pesticide, DDT, which can inhibit GJIC at a lower concentration. DDT completely inhibits the GJIC of WB-f344 at 80 lM (equal to 28.36 mg/l) (Masten et al., 2001) and the GJIC of bovine oviductal cells at 64 lM (equal to 22.7 mg/l) (Tiemann and Po¨hland, 1999). The present study revealed that GJIC inhibited by diazinon, parathion and methyl-parathion could be resumed in a time-dependent manner (p < 0.05) (Fig. 4) after removal of the target OPs. It also indirectly verified that the inhibition of GJIC function did not result from cell deaths. Other researchers also reported the recovery of GJIC inhibited by PAHs and DDT (Ghoshal et al., 1999; Herner et al., 2001; Masten et al., 2001). Although proteasomal degradation of protein kinase C is one mechanism of recovery of GJIC (Leithe et al., 2003), the recovery mechanism of GJIC inhibited by OPs remained unclear. The oxon forms of OPs were more potent acetylcholinesterase inhibitors than the parent forms (USEPA, 2001). However, our study indicated that oxons were less toxic than the parent forms evaluated with GJIC assay. This may be due to the fact that oxons are easily hydrolyzed in mammalian systems (USEPA, 2001). It was argued that hydrolytic enzymes have very important roles in the transfer of certain functional groups (i.e. P–O) to endogenous substrates (i.e. glutathione) which results in detoxification of toxins (Jokanovic´, 2001). In addition, carboxylesterase and A-esterase play important roles in detoxification after exposure to oxons (Pond et al., 1998; Tang and Chambers,
2061
1999; Karanth and Cope, 2000) and high activities A-esterase was detected in rat liver mitochondria and microsome, which hydrolyzes oxons (i.e. paraoxon and chlorpyrisoxon) to their constituent acids and alcohols (Pond et al., 1995). Ozonation byproducts of OPs had no inhibition effect on GJIC. Masten et al. (2001) also stated that mixtures of ozonation products from malathion did not inhibit GJIC and that ozonation byproducts of DDT were less inhibitory to GJIC than DDT. These findings indicated that ozonation treatment could definitely detoxify some toxicants. Since ozonation byproducts of the tested OPs were non-cytotoxic and did not inhibit GJIC, the ozonation treatment of drinking water and food crops containing the tested OPs will not form epigenetic toxicity as determined by GJIC. In summary, the present study has shown that OPs (parathion, methyl-parathion and diazinon) reversibly and concentration-dependently (p < 0.05) inhibited GJIC over a concentration range of 50–350 mg/l, while oxons (paraoxon, methyl-paraoxon and diazinon oxon) and ozonation byproduct mixtures of OPs did not inhibit GJIC at any concentrations tested under the same experiment conditions. Since there is correlation between chemical and its structure, it might be extrapolated that ozonation treatment could be applied for the detoxification of drinking water and food crops contaminated with OPs other than the tested diazinon, parathion and methyl-parathion without the formation of GJIC toxicity. Acknowledgements The financial supports from the Guangdong Provincial Natural Science Foundation of China (No. 04009780) and The Hong Kong Polytechnic University – Zhongshan University Joint Research Grant, and PolyU Research Grant A-PD94 are gratefully acknowledged. The authors would like to thank PoSeng Technology (H.K.) Co. Ltd. for providing ozone generator and Professor Jianguo He for his support on inverted fluorescence microscope. The authors would also like to gratefully acknowledge Dr. Anna Leung for proofreading of the manuscript. References Bai, Y., Ling, Z., Wang, J., 2006. Organophosphorus pesticide residues in market foods in Shaanxi area, China. Food Chem. 98 (2), 240–242. Benitez, F.J., Acero, J.L., Real, F.J., 2002. Degradation of carbofuran by using ozone, UV radiation and advanced oxidation process. J. Hazard Mater. B89, 51–65. Błasiak, J., Jaloszynski, P., Trzeciak, A., Szyfter, K., 1999. In vitro studies on the genotoxicity of the organophosphorus insecticide malathion and its two analogues. Mutat. Res. 445, 275–283. Borenfreund, E., Puerner, J.A., 1985. Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicol. Lett. 24, 119–124. Carlson, K., Ehrich, M., 1999. Organophosphorus compound-induced modification of SH-SY5Y human neuroblastoma mitochondrial transmembrane potential. Toxicol. Appl. Pharm. 160, 33–42.
2062
J. Wu et al. / Food and Chemical Toxicology 45 (2007) 2057–2063
Chiron, S., Rodriguez, A., Fernandez-Alba, A., 1998. Application of gas and liquid chromatography-mass spectrometry to the evaluation of pirimiphos methyl degradation products in industrial water under ozone treatment. J. Chromatogr. A 823, 97–107. Chu, J.Y., Wu, C.D., Chen, W.J., Chen, Z.G., 2002. Ozone Technique and Application. Chemical Industry Press, Beijing, pp. 242–277 (In Chinese). Das, P., John, G., 1999. Induction of sister chromatid exchanges and chromosome aberrations in vivo in Etroplus suratensis (Bloch) following exposure to organophosphorus pesticides. Toxicol. Lett. 104, 111– 116. El-Fouly, Mohamed H., Trosko, J.E., Chang, C.C., 1987. Scrape-loading and dye transfer: a rapid simple technique to study gap junctional intercellular communication. Exp. Cell Res. 168, 422–430. Forman, S., Ka’s, J., Fini, F., Steinberg, M., Ruml, T., 1999. The effect of different solvents on the ATP/ADP content and growth properties of Hela cells. J. Biochem. Mol. Toxicol. 13, 11–15. Galindo-Reyes, G., Fossato, V.U., Villagrana-Lizarraga, C., Dolci, F., 1999. Pesticides in water, sediments, and shrimp from a coastal lagoon off the Gulf of California. Mar. Pollut. Bull. 38 (9), 837–841. Ghoshal, S., Weber, W.J., Rummel, A.M., Trosko, J.E., Upham, B.L., 1999. Epigenetic toxicity of a mixture of polycyclic aromatic hydrocarbons on gap junctional intercellular communication before and after biodegradation. Environ. Sci. Technol. 33, 1044–1050. Gottschalk, C., Libra, J.A., Squpe, A., 2000. Ozonation of Water and Waste Water: A Practical Guide to Understanding Ozone and its Application. WILEY-VCH Verlag Gmbh, Weinheim, p. 26. Gupta, P.K., 2004. Pesticide exposure—Indian scene. Toxicology 198, 83– 90. Guzel-Seydim, Z.B., Greene, A.K., Seydim, A.G., 2004. Use of ozone in the food industry. Lebensm.-Wiss Technol. 37, 453–460. Herner, H.A., Trosko, J.E., Masten, S.J., 2001. The epigenetic toxicity of pyrene and related ozonation byproducts containing an aldehyde functional group. Environ. Sci. Technol. 35, 3576–3583. Hura, C., Leanca, M., Rusu, L., Hura, B.A., 1999. Risk assessment of pollution with pesticides in food in the Eastern Romania area (1996– 1997). Toxicol. Lett. 107, 103–107. Hwang, E.S., Cash, J.N., ZabiK, M.J., 2001. Postharvest treatments for the reduction of Mancozeb in fresh apples. J. Agr. Food Chem. 49, 3127–3132. Jokanovic´, M., 2001. Biotransformation of organophosphorus compounds. Toxicology 166, 39–160. Kang, H.G., Jeong, S.H., Cho, J.H., Kim, D.G., Park, J.M., Cho, M.H., 2004. Chlropyrifos-methyl shows anti-androgenic activity without estrogenic activity in rats. Toxicology 199, 219–230. Karanth, S., Cope, C., 2000. Carboxylesterase and A-esterase activities during maturation and aging: relationship to the toxicity of chlorpyrifos and parathion in rats. Toxicol. Sci. 58, 282–289. Ku, Y., Lin, H.S., 2002. Decomposition of phorate in aqueous solution by photolytic ozonation. Water Res. 36 (16), 4155–4159. Ku, Y., Chang, J.L., Shen, Y.S., Lin, S.Y., 1998. Decomposition of diazinon in aqueous solution by ozonation. Water Res. 32 (6), 1957– 1963. Leithe, E., Cruciani, V., Sanner, T., Mikalsen, S.O., Rivedal, E., 2003. Recovery of gap junctional intercellular communication after phorbol ester treatment requires proteasomal degradation of protein kinase C. Carcinogenesis 24 (7), 1239–1245. Luster-Teasley, S.L., Yao, J.J., Herner, H.H., Trosko, J.E., Masten, S.J., 2002. Ozonation of chrysene: evaluation of byproduct mixtures and identification of toxic constituent. Environ. Sci. Technol. 36, 869– 876. Masten, S.J., Tian, M., Upham, B.L., Trosko, J.E., 2001. Effect of selected pesticides and their ozonation by-products on gap junctional intercellular communication using rat liver epithelial cell lines. Chemosphere 44, 457–465. Navarro, H.A., Basta, P.V., Seidler, F.J., Slotkin, T.A., 2001. Neonatal chlorpyrifos administration elicits deficits in immune function in adulthood: a neural effect? Dev. Brain Res. 130, 249–252.
Okamura, A., Kamijima, M., Shibata, E., Ohtani, K., Takagi, K., Ueyama, J., Watanabe, Y., Omura, M., Wang, H., Ichihara, G., Kondo, T., Nakajima, T., 2005. A comprehensive evaluation of the testicular toxicity of dichlorvos in Wistar rats. Toxicology 213, 129– 137. Ong, K.C., Cash, J.N., Zabik, M.J., Siddiq, M., Jones, A.L., 1996. Chorine and ozone washes for pesticide removal from apples and processed apple sauce. Food Chem. 55 (2), 153–160. Pond, A.L., Chambers, H.W., Chambers, J.E., 1995. Organophosphate detoxication potential of various rat tissues via A-esterase and aliesterase activities. Toxicol. Lett. 78, 245–252. Pond, A.L., Chambers, H.W., Coyne, C.P., Chambers, J.E., 1998. Purification of two Rat hepatic proteins with A-Esterase activity toward chlorpyrifos-oxon and paraoxon. J. Pharmacol. Exp. Ther. 286, 1404–1411. Pope, C., Karanth, S., Liu, J., 2005. Pharmacology and toxicology of cholinesterase inhibitors: uses and misuses of a common mechanism of action. Environ. Toxicol. Pharmacol. 19, 433–446. Rahman, M.F., Mahboob, M., Danadevi, K., Saleha Banu, B., Grover, P., 2002. Assessment of genotoxic effects of chloropyriphos and acephate by the comet assay in mice leucocytes. Mutat. Res. 516, 139– 147. Rosenkranz, H.S., Pollack, N., Cunningham, A.R., 2000. Exploring the relationship between the inhibition of gap junctional of intercellular communication and other biological phenomena. Carcinogensis 21 (5), 1007–1011. Tang, J., Chambers, J.E., 1999. Detoxication of paraoxon by rat liver homogenate and serum carboxylesterases and A-esterases. J. Biochem. Mol. Toxicol. 13 (5), 261–268. Tariq, M.I., Afzal, S., Hussain, I., 2004. Pesticides in shallow groundwater of Bahawalnagar, Muzafargarh, D.G. Khan and Rajan Pur districts of Punjab, Pakistan. Environ. Int. 30, 471–479. Thurman, E.M., Meyer, M.T., 1996. Herbicide degradates in surface water and groundwater: introduction and overview. In: Herbicide metabolites in surface water and groundwater ACS symposium series, vol. 630, pp. 1–15. Tiemann, U., Po¨hland, R., 1999. Inhibitory effects of organochlorine pesticides on intercellular transfer of Lucifer yellow in cultured bovine oviductal cells. Reprod Toxicol. 13 (2), 123–130. Tiemann, U., Po¨hland, R., Richter, W., Wollenhaupt, K., 2002. Influence of tris(4-chlorophenyl)methanol (TCPM) on gap junction-mediated intercellular communication of cultured bovine granulose cells. Toxicol. In Vitro 16, 129–139. Trosko, J.E., Chang, C.C., Upham, B., Wilson, M., 1998. Epigenetic toxicology as toxicant-induced changes in intracellular signalling leading to altered gap junctional intercellular communication. Toxicol. Lett. 71 (8), 102–103. Upham, B.L., Yao, J.J., Trosko, J.E., Masten, S.J., 1995. Determination of the efficacy of ozone treatment systems using a gap junction intercellular communication bioassay. Environ. Sci. Technol. 29, 2923–2928. Upham, B.L., Weis, L.M., Rummel, A.M., Masten, S.J., Trosko, J.E., 1996. The effects of anthracene and methylated anthracenes on gap junctional intercellular communication in rat liver epithelial cells. Fund. Appl. Toxicol. 34, 260–264. Upham, B.L., Weis, L.M., Trosko, J.E., 1998. Modulated gap junctional intercellular communication as a biomarker of PAH epigenetic toxicity: structure–function relationship. Environ. Health Persp. 106, 975–981. United States Environmental Protection Agency (USEPA), (2001). The Incorporation of water treatment effects on pesticide removal and transformation in food quality protection act (FQPA) drinking water assessments. Officer of Pesticide Programs, Washington, DC 20460, p. 35. Wagner, E.D., McMillan, S.M., Plewa, M.J., 2005. Cytotoxicity of organophosphorus ester (OP) insecticides and cytotoxic synergism of 2-Acetoxyacetylaminofluorene (2AAAF) in Chinese Hamster Ovary (CHO) cells. Bull. Environ. Contam. Toxicol. 75, 329–334.
J. Wu et al. / Food and Chemical Toxicology 45 (2007) 2057–2063 Wu, J.G., Luan, T.G., Lan, C.Y., Lo, T.W.H., Chan, G.Y.S., 2007. Removal of residual pesticides on vegetable using ozonated water. Food Control 18 (5), 466–472. Xu, X.H., Zhao, W.R., 2003. Ozonation of Water and Wasterwater. Chemical Industry Press, Beijing, pp. 363–393 (In Chinese).
2063
Yeh, S.P., Sung, T.G., Chang, C.C., Cheng, W., Kuo, C.M., 2005. Effects of an organophosphorus insecticide, trichlorfon, on hematological parameters of the giant freshwater prawn, Macrobrachium rosenbergii (de Man). Aquaculture 243, 383–392.
Effects of organophosphorus pesticides and their ozonation byproducts on gap junctional intercellular communication in rat liver cell line Jiguo Wu a
a,c
, Li Lin a, Tiangang Luan
a,*
, Yuk Sing Chan Gilbert b, Chongyu Lan
a,*
State Key Laboratory of Biocontrol, School of Life Sciences, Zhongshan (Sun Yat-sen) University, Guangzhou 510275, PR China b Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hong Kong, PR China c School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, PR China Received 15 June 2006; accepted 10 May 2007
Abstract The effects of organophosphorus pesticides (OPs), oxons and their ozonation byproducts on gap junctional intercellular communication (GJIC) on cultured BRL cell line were investigated using scrape loading and dye transfer (SL/DT) technique. The neutral red uptake assay was used to identify the non-cytotoxic levels of diazinon, parathion and methyl-parathion applied to GJIC assay. The concentration-dependent inhibition of GJIC was observed over a range of 50–350 mg/l diazinon, parathion and methyl-parathion after 90 min incubation compared with the vehicle control. However, oxons and ozonation byproducts of OPs had no inhibition effect on GJIC at any of the concentrations tested. The inhibition of GJIC by OPs was reversible after removal of the tested pesticides followed by incubation with fresh medium. The present study suggested that the ozonation treatment could be used for the detoxification of drinking water and food crops contaminated with diazinon, parathion and methyl-parathion without formation of GJIC toxicity. 2007 Elsevier Ltd. All rights reserved. Keywords: Diazinon; Parathion; Methyl-parathion; Ozone; Buffalo rat liver
1. Introduction Organophosphorus pesticides (OPs) are one of the most important groups of insecticides and are widely applied to pest control in agriculture and hygiene. The extensive application of OPs can subsequently release OPs into hydrological systems (Thurman and Meyer, 1996; Galindo-Reyes et al., 1999; Tariq et al., 2004) or contaminate food crops (Hura et al., 1999; Gupta, 2004; Bai et al., 2006), which may pose a seriously adverse impact to non-
Abbreviations: Buffalo rat liver (BRL); Dulbecco’s Modified Eagle Medium (DMEM); Gap junctional intercellular communication (GJIC); Organophosphorus pesticide (OP); Phosphate buffered saline (PBS); Scrape loading and dye transfer (SL/DT). * Corresponding authors. Tel.: +86 20 84036296; fax: +86 20 84113652. E-mail address: [email protected] (T. Luan). 0278-6915/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2007.05.006
target organisms and humans due to various toxicities of OPs. OPs elicit their extensive toxicities through various mechanisms and pathways. Primarily, OPs elicit their acute toxicities through a common mechanism of inhibition of acetylcholinesterase, ultimately modifying cholinergic signaling through disruption of acetylcholine degradation (Pope et al., 2005). OPs also elicit their toxicities via other mechanisms including cytotoxicity (Carlson and Ehrich, 1999; Wagner et al., 2005), genotoxicity (Błasiak et al., 1999; Das and John, 1999; Rahman et al., 2002), disruption of sex hormone and reproductivity (Kang et al., 2004; Okamura et al., 2005), and immunotoxicity (Navarro et al., 2001; Yeh et al., 2005). Gap junctional intercellular communication (GJIC) has been considered as an important determinant for normal cell growth, development, differentiation as well as
2058
J. Wu et al. / Food and Chemical Toxicology 45 (2007) 2057–2063
maintenance of homeostasis in multicellular organisms. Inhibition of GJIC by various chemicals has been postulated to be a factor in the tumor promotion phase of carcinogenesis, teratogenesis, neurotoxicity, reproductive dysfunction and other chemically-induced disease states (El-Fouly et al., 1987). Untimely or chronic disruption of GJIC during embryonic or fetal development could lead to embryonic lethality or teratogenesis and chemical modulation of GJIC in initiated tissues has been shown to lead to tumor promotion (Trosko et al., 1998). GJIC assay has been shown to be a reliable, simple, inexpensive in vitro assay as well as a good predictor of epigenetic toxicants (Rosenkranz et al., 2000), therefore it has been a useful marker for testing the epigenetic toxicity of some toxicants such as polycyclic aromatic hydrocarbons (PAHs), organochlorine compounds (Upham et al., 1996, 1998; Tiemann and Po¨hland, 1999; Tiemann et al., 2002) and the ozonation byproducts or biometabolites of the parent compounds (Upham et al., 1995; Ghoshal et al., 1999; Herner et al., 2001; Masten et al., 2001; Luster-Teasley et al., 2002). Ozone was widely used in drinking water treatment for disinfection and oxidation of contaminant. Ozonation treatment can be used to oxidize OPs in aqueous solution (Chiron et al., 1998; Ku et al., 1998; Masten et al., 2001; Benitez et al., 2002; Ku and Lin, 2002). Ozone has also been applied to remove residual pesticides in food crops (Ong et al., 1996; Hwang et al., 2001; Wu et al., 2007) as well as food hygiene and sanitation (Guzel-Seydim et al., 2004). However, the target compounds will not be mineralized, but transformed to metabolites of lesser molecular weight in actual ozone application (Gottschalk et al., 2000). So toxicity assessments of both parent OPs and their ozonation byproducts are necessary for the sake of safety consideration after ozonation treatment. The objective of the present study was (1) to investigate the effects of OPs and their ozonation byproducts on GJIC; and (2) to evaluate the safety of ozonation treatment of OPs using GJIC bioassay. 2. Methods and materials 2.1. Chemicals and reagents The pesticide standards of diazinon (CAS#: 333-41-5; purity: 98.1%), parathion (CAS#: 56-38-2; purity: 98%), paraoxon (CAS#: 311-45-5; purity: 98.6%), methyl-parathion (CAS#: 298-00-0; purity: 99.9%) and methyl-paraoxon (CAS#: 950-35-6; purity: 99.0%) were from Supelco, USA. Diazinon oxon (CAS#: 962-58-3; purity: 99.0%) was from Wako, Japan. All pesticide standard stock solutions (20 mg/ml) were prepared in acetone. Analytical grade acetone and ethyl acetate (CAS#: 141-78-6) (Guangzhou Chemical Factory, China) were redistilled before use. Neutral red was from Tianjing Chemical Development Center, China. Trypsin solution (0.25%) was from GIBCO, USA. Lucifer yellow CH was from Invitrogen, USA, and dissolved in phosphate buffered saline (PBS) (pH 7.2) with concentration of 1 mg/ml.
2.2. Preparation of the ozonation byproducts of OPs Ozonation experiments were carried out by bubbling gaseous ozone of 5.5 mg/l at a flow rate of 90 ml/min into a 250 ml glass column through a
disperser at the column bottom for preparation of ozonation byproducts of the target OPs. Two hundred ml of individual OP solution (initial concentration: 200 lg/ml) was ozonated for various time periods. The excess gaseous ozone was trapped in 2% potassium iodide (KI) solution. After ozonation of diazinon and parathion solution for 180 min and methyl-parathion solution for 210 min (more than 99% of parent pesticides was degraded (data not shown)), 100 ml samples were collected and 99.999% purity nitrogen was bubbled into the samples for 2 min to quench the reactions between ozone and pesticides. The samples were transferred to 250 ml separated funnels and extracted three times with 100 ml ethyl acetate. The combined extracts were evaporated to approximately 5 ml by the rotary evaporator (BUCHI, Switzerland) followed by purging of the remaining ethyl acetate with high purity nitrogen, then the residual was reconstituted in 0.5 ml acetone. The control sample was prepared with ultra-pure water (Millipore system, USA) without addition of pesticide according to the same procedure. The parent pesticide concentration was used as the basis to calculate the concentration of ozonation byproducts.
2.3. Cell culture The buffalo rat liver (BRL) cell line from Experimental Animal Center of Sun Yat-sen University were grown in Dulbecco’s Modified Eagle Medium (DMEM) (GIBCO, USA), supplemented with 5% newborn calf serum (Sijiqing, Hangzhou, China), 100 U/ml penicillin and streptomycin 100 lg/ml and incubated at 37 C in a humidified atmosphere containing 5% (v/v) CO2 and 95% air. The cells were grown in 25 mm2 tissue culture flasks or tissue culture dishes of 40 · 10 mm (TPP, Europe/Switzerland) or 96-well plates (TPP, Europe/Switzerland). The cell cultures were subcultured in fresh DMEM every three days.
2.4. Neutral red uptake assay The cytotoxicities of the OPs and their ozonation byproducts were tested by neutral red uptake assay developed by Borenfreund and Puerner (1985) and modified by Masten et al. (2001). The procedure is briefly described as follows: Neutral red dye stock solution (0.4%) was prepared in PBS and filtered with 0.22 lm syringe-filter (TPP, Europe/Switzerland). This neutral red solution was diluted to a final working concentration of 0.04% in DMEM. The diluted solution was incubated at 37 C overnight followed by filtration with 0.22 lm syringe-filter to remove fine precipitates. Cells were cultured in 96-well plates for two days. After incubation with DMEM containing various concentrations of OPs or the ozonation byproducts or vehicle control for 3 h, the cells were rinsed three times with PBS and incubated with DMEM containing 0.04% neutral red dye for 3 h. Then the medium was discarded and the cells were rinsed three times with PBS. Subsequently, 0.2 ml solution containing 1% acetic acid and 50% ethanol was added to each well of the 96-well plates to extract neutral red dye for 20 min. Wellscan MK3 (Labsystems Dragon, Finland) was used to determine the absorbance of neutral red dye released from the cells in each well of the 96-well plates at a wavelength of 550 nm. Every treatment was repeated four times. The cytotoxicities of the target pesticides and their ozonation byproducts were reported as relative viability to the control.
2.5. GJIC assay GJIC assay was carried out in 40 · 10 mm tissue culture dishes with 100% confluent monolayer cells grown in two ml DMEM supplemented with 5% newborn calf serum, 100 U/ml penicillin and streptomycin 100 lg/ml. GJIC was detected using the scrape loading and dye transfer (SL/DT) technique developed by El-Fouly et al. (1987). Assays for different treatments and vehicle control were run in triplicate tissue culture dishes. Monolayer cells with 100% confluence were incubated with target compounds for a constant time period with different concentrations or for various time periods with constant concentration. After exposure to the target compounds, the cells were rinsed three times with PBS and one ml of lucifer yellow CH solution was added to the cell cultures and scrape-
J. Wu et al. / Food and Chemical Toxicology 45 (2007) 2057–2063 Table 1 The evaluation criterion of inhibitory GJIC
a
Relative migration to the control (%)
Response
Reference
<30
Complete inhibition Partial inhibition No inhibition
Herner et al. (2001)
30–90 >90
2059
120 100 80 60
Diazinon Methyl parathion Parathion
40 20 0
b
120
Relative viability
0 loaded with several scrapes using a steel surgical blade (El-Fouly et al., 1987). The dye solution was left on the cell cultures for 3 min, then discarded and the cell cultures were carefully rinsed three times with PBS to remove detached cells and background fluorescence. Several drops of 4% formalin in PBS were added to fix the cell cultures. An inverted fluorescence microscope equipped with a digital camera (Nikon Eclipse TE 2000U system, Nikon ACT-1 version 2.62, Nikon corporation, Japan) was used to record the migration of the lucifer yellow CH dye from the edge cells of the scrape. The migration was measured on the micrograph. An average value of 30 measurements for each treatment (10 measurements per dish) was regarded as the migration of the dye in the cell cultures. The percentage of the migration of the dye in the cell cultures exposed to target compounds to the migration which the dye traveled in the vehicle control was used to evaluate the inhibition of GJIC. Table 1 shows the evaluation criterion of inhibitory GJIC proposed by Herner et al. (2001).
100
50
The cells were cultured under the same conditions as the GJIC assay and incubated in DMEM containing the target pesticides or ozonation byproducts with constant concentration for 90 min. After removing the target compounds, the cells were rinsed three times with PBS and incubated in fresh DMEM for 30, 60, 90, 120, 150 min, respectively. These cell cultures were then used to determine GJIC function according to the SL/ DT technique described in GJIC assay. Every treatment was conducted in triplicate.
150
200
250
300
350
400
200
250
300
350
400
300
350
400
80 60
Diazinon oxon Methyl paraoxon Methyl paraoxon
40 20 0
c
120
0
50
100
150
100 80 60 Diazinon byproducts Methyl parathion byproducts Parathion byproducts
40
2.6. GJIC recovery test
100
20 0 0
50
100
150
200
250
(mg/ml) Fig. 1. Concentration effects on cytotoxicity. The cell cultures were exposed to (a) parent pesticides diazinon, methyl-parathion and methylparathion; (b) oxons (diazinon oxon, methyl-paraoxon and paraoxon); (c) ozonation byproducts of diazinon, methyl-parathion and parathion for 3 h. The error bars were one standard deviation (n = 4).
2.7. Data analysis The results were reported as a mean ±1 SD. A t-test was performed to determine whether different treatments differed significantly from the control at p 6 0.05, using the software SAS (SAS Institute Inc., Cary, NC, USA.).
3. Results 3.1. Cytotoxicity assay In order to identify the non-cytotoxic levels of the selected OPs applied to GJIC studies, neutral red uptake assay was used to determine the cytotoxicity of the target compounds. Diazinon, parathion and methyl-parathion were non-cytotoxic at the tested levels (10–350 mg/l) after incubation for 3 h (Fig. 1a). And diazinon oxon, paraoxon and methyl-paraoxon were also non-cytotoxic at concentrations of 50–400 mg/l (Fig. 1b). Fig. 1c also indicates that the ozonation byproducts were non-cytotoxic for all of the concentrations tested (50–400 mg/l). 3.2. Concentration effect on GJIC For evaluation of concentration response, GJIC levels were detected after the cells were exposed to the target
compounds with non-cytotoxic levels for 90 min. Methylparathion inhibited GJIC over a concentration range of 150–300 mg/l (Fig. 2a) and partially inhibited GJIC was found (84.4% to the control) at the concentration of 150 mg/l. Almost completely inhibited GJIC was found at 300 mg/l (32.4% to the control). The results indicated that GJIC was inhibited by methyl-parathion in a concentration dependent manner (p < 0.05), beginning at 150 mg/ l. However, methyl-paraoxon and ozonation byproducts of methyl-parathion did not inhibit GJIC at any of the concentrations tested (p > 0.05). Parathion concentration-dependent (p < 0.05) inhibited GJIC over a concentration range of 100–350 mg/l (Fig. 2b). GJIC was partially inhibited at a concentration of 100 mg/l (88.8% to the control); and almost completely inhibited at concentration of 350 mg/l (32% to the control). However, paraoxon and ozonation byproducts of parathion did not inhibit GJIC at any of the concentrations tested (p > 0.05). Diazinon inhibited GJIC over a concentration range of 50–300 mg/l, while diazinon oxon and ozonation byproducts of diazinon did not inhibit GJIC at any of the concentrations tested (Fig. 2c). GJIC was also inhibited by diazinon at a concentration dependent manner (p < 0.05).
2060
120
120 100 80 60 40 20 0
100
Methyl parathion Methyl paraoxon Methyl parathion byproducts
0
50
100
150
200
250
300
350
400
Relative GJIC inhibition
b 120 100 80 60 40 20 0
c
120
Relative GJIC inhibition
a
J. Wu et al. / Food and Chemical Toxicology 45 (2007) 2057–2063
80 60 40 Diazinon Methyl parathion Parathion
20 Parathion Paraoxon Parathion byproducts
0
50
100
150
0 0 200
250
300
350
400
100 80
20
40
60
80 100 120 140 Time (min)
160 180
Fig. 3. Time effects of OPs on GJIC. The cell cultures were incubated for various time periods with DMEM containing 250 mg/l diazinon, methylparathion and parathion. The error bars were one standard deviation (n = 30).
60 40
110
Diazinon Diazinon oxon Diazinon byproducts
0 0
50
100
150
Diazinon Methyl parathion Parathion
100 200
250
300
350
400
(mg/ml) Fig. 2. Concentration effects on GJIC. Cell cultures were exposed to (a) methyl-parathion, methyl-paraoxon and ozonation byproducts of methylparathion; (b) parathion, paraoxon and ozonation byproducts of parathon; (c) diazinon, diazinon oxon and ozonation byproducts of diazinon for 90 min. The error bars were one standard deviation (n = 30).
Relative GJIC inhibition
20
90 80 70 60 50 40 30
Diazinon partially inhibited GJIC at a concentration of 50 mg/l (89.4% to the control) and almost completely inhibited GJIC at a concentration of 300 mg/l (30.4% to the control). These results suggested that the tested pesticides methylparathion, parathion and diazinon inhibited GJIC in a concentration-dependent manner, while their ozonation byproducts did not inhibit GJIC in BRL cell line.
20 -20
0
20
40
60 80 100 Time (min)
120
140
160
Fig. 4. Time of recoveries for OPs inhibitory to GJIC. The cell cultures were preincubated with DMEM containing 300 mg/l diazinon, methylparathion and 350 mg/l parathion for 90 min. The error bars were one standard deviations (n = 30).
3.3. Time effect on GJIC
methyl-parathion and parathion were recovered to 91.3%, 84.7% and 92.7%, respectively.
During all treatment times, the inhibition of GJIC was the greatest at 90 min for methyl-parathion, parathion and diazinon (Fig. 3).
4. Discussion
3.4. Recovery of GJIC The inhibition of GJIC by the tested pesticides could be time-dependent (p < 0.05) recovered after removal of the toxicants (Fig. 4). When the cells pretreated with target toxicants were incubated in the fresh medium for 150 min, the function of GJIC inhibited by diazinon,
To evaluate the epigenetic toxicity of OPs and the relevant ozonation byproducts, individual OP was ozonated and the toxicity of the mixture of byproducts produced was determined using GJIC assay in BRL cell line. As a result of ozonation of diazinon and parathion solution for 180 min and methyl-parathion solution for 210 min, more than 99% of the parent pesticides was degraded (data not shown). Generally speaking, the ozone level of 1–3 mg/ l was applied for 5–15 min in a typical full scale water treat-
J. Wu et al. / Food and Chemical Toxicology 45 (2007) 2057–2063
ment (Chu et al., 2002; Xu and Zhao, 2003), depending on the properties of water source. In nearly all cases, the target compounds will not be mineralized by ozone, but transformed to intermediates, and some of the products formed do not react further with ozone (Gottschalk et al., 2000). Therefore, the ozonation byproduct components will not be largely changed for the prolonged contact time and high initial concentration of the parent pesticide. The ozonation byproducts were extracted with ethyl acetate and reconstituted in vehicle solvent acetone which was less toxic than dimethyl sulfoxide (DMSO), the most widely used solvent in bioassay systems (Forman et al., 1999). The concentrations of the ozonation byproducts were calculated according to those of the parent pesticides. Since ozonation of organophosphorus pesticides containing P@S bond can form trace oxons as primary byproducts (Wu et al., 2007), oxons were singled out to test their toxicity. GJIC in the BRL cell line was inhibited by the selected OPs in a concentration dependent manner (p < 0.05) over the range of 50–350 mg/l. The neutral red uptake assay supported that the inhibition effect of OPs on GJIC in the BRL cell line did not result from the death of the cells. The pesticide levels at which the GJIC was most completely inhibited were 300 mg/l for methyl-parathion and diazinon (Fig. 2a and c) and 350 mg/l for parathion ( Fig. 2b). These levels were similar to that of malathion (1000 lM, equal to 330.36 mg/l), at which GJIC in BW-f343 cell line was completely inhibited (Masten et al., 2001). However, these levels of the tested pesticides were higher than that of organochlorine pesticide, DDT, which can inhibit GJIC at a lower concentration. DDT completely inhibits the GJIC of WB-f344 at 80 lM (equal to 28.36 mg/l) (Masten et al., 2001) and the GJIC of bovine oviductal cells at 64 lM (equal to 22.7 mg/l) (Tiemann and Po¨hland, 1999). The present study revealed that GJIC inhibited by diazinon, parathion and methyl-parathion could be resumed in a time-dependent manner (p < 0.05) (Fig. 4) after removal of the target OPs. It also indirectly verified that the inhibition of GJIC function did not result from cell deaths. Other researchers also reported the recovery of GJIC inhibited by PAHs and DDT (Ghoshal et al., 1999; Herner et al., 2001; Masten et al., 2001). Although proteasomal degradation of protein kinase C is one mechanism of recovery of GJIC (Leithe et al., 2003), the recovery mechanism of GJIC inhibited by OPs remained unclear. The oxon forms of OPs were more potent acetylcholinesterase inhibitors than the parent forms (USEPA, 2001). However, our study indicated that oxons were less toxic than the parent forms evaluated with GJIC assay. This may be due to the fact that oxons are easily hydrolyzed in mammalian systems (USEPA, 2001). It was argued that hydrolytic enzymes have very important roles in the transfer of certain functional groups (i.e. P–O) to endogenous substrates (i.e. glutathione) which results in detoxification of toxins (Jokanovic´, 2001). In addition, carboxylesterase and A-esterase play important roles in detoxification after exposure to oxons (Pond et al., 1998; Tang and Chambers,
2061
1999; Karanth and Cope, 2000) and high activities A-esterase was detected in rat liver mitochondria and microsome, which hydrolyzes oxons (i.e. paraoxon and chlorpyrisoxon) to their constituent acids and alcohols (Pond et al., 1995). Ozonation byproducts of OPs had no inhibition effect on GJIC. Masten et al. (2001) also stated that mixtures of ozonation products from malathion did not inhibit GJIC and that ozonation byproducts of DDT were less inhibitory to GJIC than DDT. These findings indicated that ozonation treatment could definitely detoxify some toxicants. Since ozonation byproducts of the tested OPs were non-cytotoxic and did not inhibit GJIC, the ozonation treatment of drinking water and food crops containing the tested OPs will not form epigenetic toxicity as determined by GJIC. In summary, the present study has shown that OPs (parathion, methyl-parathion and diazinon) reversibly and concentration-dependently (p < 0.05) inhibited GJIC over a concentration range of 50–350 mg/l, while oxons (paraoxon, methyl-paraoxon and diazinon oxon) and ozonation byproduct mixtures of OPs did not inhibit GJIC at any concentrations tested under the same experiment conditions. Since there is correlation between chemical and its structure, it might be extrapolated that ozonation treatment could be applied for the detoxification of drinking water and food crops contaminated with OPs other than the tested diazinon, parathion and methyl-parathion without the formation of GJIC toxicity. Acknowledgements The financial supports from the Guangdong Provincial Natural Science Foundation of China (No. 04009780) and The Hong Kong Polytechnic University – Zhongshan University Joint Research Grant, and PolyU Research Grant A-PD94 are gratefully acknowledged. The authors would like to thank PoSeng Technology (H.K.) Co. Ltd. for providing ozone generator and Professor Jianguo He for his support on inverted fluorescence microscope. The authors would also like to gratefully acknowledge Dr. Anna Leung for proofreading of the manuscript. References Bai, Y., Ling, Z., Wang, J., 2006. Organophosphorus pesticide residues in market foods in Shaanxi area, China. Food Chem. 98 (2), 240–242. Benitez, F.J., Acero, J.L., Real, F.J., 2002. Degradation of carbofuran by using ozone, UV radiation and advanced oxidation process. J. Hazard Mater. B89, 51–65. Błasiak, J., Jaloszynski, P., Trzeciak, A., Szyfter, K., 1999. In vitro studies on the genotoxicity of the organophosphorus insecticide malathion and its two analogues. Mutat. Res. 445, 275–283. Borenfreund, E., Puerner, J.A., 1985. Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicol. Lett. 24, 119–124. Carlson, K., Ehrich, M., 1999. Organophosphorus compound-induced modification of SH-SY5Y human neuroblastoma mitochondrial transmembrane potential. Toxicol. Appl. Pharm. 160, 33–42.
2062
J. Wu et al. / Food and Chemical Toxicology 45 (2007) 2057–2063
Chiron, S., Rodriguez, A., Fernandez-Alba, A., 1998. Application of gas and liquid chromatography-mass spectrometry to the evaluation of pirimiphos methyl degradation products in industrial water under ozone treatment. J. Chromatogr. A 823, 97–107. Chu, J.Y., Wu, C.D., Chen, W.J., Chen, Z.G., 2002. Ozone Technique and Application. Chemical Industry Press, Beijing, pp. 242–277 (In Chinese). Das, P., John, G., 1999. Induction of sister chromatid exchanges and chromosome aberrations in vivo in Etroplus suratensis (Bloch) following exposure to organophosphorus pesticides. Toxicol. Lett. 104, 111– 116. El-Fouly, Mohamed H., Trosko, J.E., Chang, C.C., 1987. Scrape-loading and dye transfer: a rapid simple technique to study gap junctional intercellular communication. Exp. Cell Res. 168, 422–430. Forman, S., Ka’s, J., Fini, F., Steinberg, M., Ruml, T., 1999. The effect of different solvents on the ATP/ADP content and growth properties of Hela cells. J. Biochem. Mol. Toxicol. 13, 11–15. Galindo-Reyes, G., Fossato, V.U., Villagrana-Lizarraga, C., Dolci, F., 1999. Pesticides in water, sediments, and shrimp from a coastal lagoon off the Gulf of California. Mar. Pollut. Bull. 38 (9), 837–841. Ghoshal, S., Weber, W.J., Rummel, A.M., Trosko, J.E., Upham, B.L., 1999. Epigenetic toxicity of a mixture of polycyclic aromatic hydrocarbons on gap junctional intercellular communication before and after biodegradation. Environ. Sci. Technol. 33, 1044–1050. Gottschalk, C., Libra, J.A., Squpe, A., 2000. Ozonation of Water and Waste Water: A Practical Guide to Understanding Ozone and its Application. WILEY-VCH Verlag Gmbh, Weinheim, p. 26. Gupta, P.K., 2004. Pesticide exposure—Indian scene. Toxicology 198, 83– 90. Guzel-Seydim, Z.B., Greene, A.K., Seydim, A.G., 2004. Use of ozone in the food industry. Lebensm.-Wiss Technol. 37, 453–460. Herner, H.A., Trosko, J.E., Masten, S.J., 2001. The epigenetic toxicity of pyrene and related ozonation byproducts containing an aldehyde functional group. Environ. Sci. Technol. 35, 3576–3583. Hura, C., Leanca, M., Rusu, L., Hura, B.A., 1999. Risk assessment of pollution with pesticides in food in the Eastern Romania area (1996– 1997). Toxicol. Lett. 107, 103–107. Hwang, E.S., Cash, J.N., ZabiK, M.J., 2001. Postharvest treatments for the reduction of Mancozeb in fresh apples. J. Agr. Food Chem. 49, 3127–3132. Jokanovic´, M., 2001. Biotransformation of organophosphorus compounds. Toxicology 166, 39–160. Kang, H.G., Jeong, S.H., Cho, J.H., Kim, D.G., Park, J.M., Cho, M.H., 2004. Chlropyrifos-methyl shows anti-androgenic activity without estrogenic activity in rats. Toxicology 199, 219–230. Karanth, S., Cope, C., 2000. Carboxylesterase and A-esterase activities during maturation and aging: relationship to the toxicity of chlorpyrifos and parathion in rats. Toxicol. Sci. 58, 282–289. Ku, Y., Lin, H.S., 2002. Decomposition of phorate in aqueous solution by photolytic ozonation. Water Res. 36 (16), 4155–4159. Ku, Y., Chang, J.L., Shen, Y.S., Lin, S.Y., 1998. Decomposition of diazinon in aqueous solution by ozonation. Water Res. 32 (6), 1957– 1963. Leithe, E., Cruciani, V., Sanner, T., Mikalsen, S.O., Rivedal, E., 2003. Recovery of gap junctional intercellular communication after phorbol ester treatment requires proteasomal degradation of protein kinase C. Carcinogenesis 24 (7), 1239–1245. Luster-Teasley, S.L., Yao, J.J., Herner, H.H., Trosko, J.E., Masten, S.J., 2002. Ozonation of chrysene: evaluation of byproduct mixtures and identification of toxic constituent. Environ. Sci. Technol. 36, 869– 876. Masten, S.J., Tian, M., Upham, B.L., Trosko, J.E., 2001. Effect of selected pesticides and their ozonation by-products on gap junctional intercellular communication using rat liver epithelial cell lines. Chemosphere 44, 457–465. Navarro, H.A., Basta, P.V., Seidler, F.J., Slotkin, T.A., 2001. Neonatal chlorpyrifos administration elicits deficits in immune function in adulthood: a neural effect? Dev. Brain Res. 130, 249–252.
Okamura, A., Kamijima, M., Shibata, E., Ohtani, K., Takagi, K., Ueyama, J., Watanabe, Y., Omura, M., Wang, H., Ichihara, G., Kondo, T., Nakajima, T., 2005. A comprehensive evaluation of the testicular toxicity of dichlorvos in Wistar rats. Toxicology 213, 129– 137. Ong, K.C., Cash, J.N., Zabik, M.J., Siddiq, M., Jones, A.L., 1996. Chorine and ozone washes for pesticide removal from apples and processed apple sauce. Food Chem. 55 (2), 153–160. Pond, A.L., Chambers, H.W., Chambers, J.E., 1995. Organophosphate detoxication potential of various rat tissues via A-esterase and aliesterase activities. Toxicol. Lett. 78, 245–252. Pond, A.L., Chambers, H.W., Coyne, C.P., Chambers, J.E., 1998. Purification of two Rat hepatic proteins with A-Esterase activity toward chlorpyrifos-oxon and paraoxon. J. Pharmacol. Exp. Ther. 286, 1404–1411. Pope, C., Karanth, S., Liu, J., 2005. Pharmacology and toxicology of cholinesterase inhibitors: uses and misuses of a common mechanism of action. Environ. Toxicol. Pharmacol. 19, 433–446. Rahman, M.F., Mahboob, M., Danadevi, K., Saleha Banu, B., Grover, P., 2002. Assessment of genotoxic effects of chloropyriphos and acephate by the comet assay in mice leucocytes. Mutat. Res. 516, 139– 147. Rosenkranz, H.S., Pollack, N., Cunningham, A.R., 2000. Exploring the relationship between the inhibition of gap junctional of intercellular communication and other biological phenomena. Carcinogensis 21 (5), 1007–1011. Tang, J., Chambers, J.E., 1999. Detoxication of paraoxon by rat liver homogenate and serum carboxylesterases and A-esterases. J. Biochem. Mol. Toxicol. 13 (5), 261–268. Tariq, M.I., Afzal, S., Hussain, I., 2004. Pesticides in shallow groundwater of Bahawalnagar, Muzafargarh, D.G. Khan and Rajan Pur districts of Punjab, Pakistan. Environ. Int. 30, 471–479. Thurman, E.M., Meyer, M.T., 1996. Herbicide degradates in surface water and groundwater: introduction and overview. In: Herbicide metabolites in surface water and groundwater ACS symposium series, vol. 630, pp. 1–15. Tiemann, U., Po¨hland, R., 1999. Inhibitory effects of organochlorine pesticides on intercellular transfer of Lucifer yellow in cultured bovine oviductal cells. Reprod Toxicol. 13 (2), 123–130. Tiemann, U., Po¨hland, R., Richter, W., Wollenhaupt, K., 2002. Influence of tris(4-chlorophenyl)methanol (TCPM) on gap junction-mediated intercellular communication of cultured bovine granulose cells. Toxicol. In Vitro 16, 129–139. Trosko, J.E., Chang, C.C., Upham, B., Wilson, M., 1998. Epigenetic toxicology as toxicant-induced changes in intracellular signalling leading to altered gap junctional intercellular communication. Toxicol. Lett. 71 (8), 102–103. Upham, B.L., Yao, J.J., Trosko, J.E., Masten, S.J., 1995. Determination of the efficacy of ozone treatment systems using a gap junction intercellular communication bioassay. Environ. Sci. Technol. 29, 2923–2928. Upham, B.L., Weis, L.M., Rummel, A.M., Masten, S.J., Trosko, J.E., 1996. The effects of anthracene and methylated anthracenes on gap junctional intercellular communication in rat liver epithelial cells. Fund. Appl. Toxicol. 34, 260–264. Upham, B.L., Weis, L.M., Trosko, J.E., 1998. Modulated gap junctional intercellular communication as a biomarker of PAH epigenetic toxicity: structure–function relationship. Environ. Health Persp. 106, 975–981. United States Environmental Protection Agency (USEPA), (2001). The Incorporation of water treatment effects on pesticide removal and transformation in food quality protection act (FQPA) drinking water assessments. Officer of Pesticide Programs, Washington, DC 20460, p. 35. Wagner, E.D., McMillan, S.M., Plewa, M.J., 2005. Cytotoxicity of organophosphorus ester (OP) insecticides and cytotoxic synergism of 2-Acetoxyacetylaminofluorene (2AAAF) in Chinese Hamster Ovary (CHO) cells. Bull. Environ. Contam. Toxicol. 75, 329–334.
J. Wu et al. / Food and Chemical Toxicology 45 (2007) 2057–2063 Wu, J.G., Luan, T.G., Lan, C.Y., Lo, T.W.H., Chan, G.Y.S., 2007. Removal of residual pesticides on vegetable using ozonated water. Food Control 18 (5), 466–472. Xu, X.H., Zhao, W.R., 2003. Ozonation of Water and Wasterwater. Chemical Industry Press, Beijing, pp. 363–393 (In Chinese).
2063
Yeh, S.P., Sung, T.G., Chang, C.C., Cheng, W., Kuo, C.M., 2005. Effects of an organophosphorus insecticide, trichlorfon, on hematological parameters of the giant freshwater prawn, Macrobrachium rosenbergii (de Man). Aquaculture 243, 383–392.