Organophosphate (OP) insecticides exhibit a high level of pest control ability combined with a relatively low degree of environmental toxicity. Hence, they are used widely around the world in agriculture and in households, which has led to a variety of negative effects in nontarget species including humans. In addition, accidental as well as suicidal poisoning cases related to these pesticides have increased over the years (1, 2). Bidrin® (BD) is a water-soluble insecticide formulation manufactured in the United States with the active ingredient dicrotophos ((E)-2-dimethylcarbamoyl-1-methylvinyl dimethyl phosphate) and is classified as a restricted-use pesticide due to its high toxicity to humans and wildlife.a Dicrotophos is used against a variety of sucking, boring, and chewing insects of crops both inside and outside the United States (Amvac Chemical Corp., 1994; EXTOXNET, 1995a).
Toxicity from BD is primarily through inhibition of acetylcholinesterase (AChE) enzyme. Human case reports indicate that acute intoxication from OP compounds can be associated with acute tubular necrosis (3). After acute human OP poisoning, greater levels of OP have been found to be localized in the kidneys than in blood (4). In rats, OP have been shown to cause acute renal tubular injury (5). Furthermore, the renal dysfunction was found not to correlate with the degree of suppression of cholinesterase. This indicated that besides the primary mechanism of impairment of neural conduction, other pathways of OP toxicity to renal proximal tubular cells might be present. The alternate mechanisms by which OP induce renal tubular cytotoxicity need to be investigated. Since reactive oxygen species (ROS) are known to mediate many toxin-induced renal tubular injuries (6,7,8,9), we examined whether ROS play a role in BD-induced renal tubular epithelial cell (LLC-PK1) toxicity. Results from this study suggest that ROS and concomitant lipid peroxidation, at least in part, are involved in the toxic effects of BD on renal cells and that oxidative stress may play a role in the structural and functional impairment caused by OP intoxication. Furthermore, this study demonstrates that antioxidants that inhibit lipid peroxidation, such as desferrioxamine (DFO) and 2-methylaminochroman (2-MAC, U83836E), can protect renal tubular cells against BD-induced damage in the cell culture setting.
Materials and Methods
LLC-PK1 cells, an analog of porcine proximal tubular epithelial cells, were obtained from American Type Culture Collection (Rockville, MD) and were cultured as reported previously (10). For experimental studies, cells were seeded in 48-well culture plates and grown to confluence. Experiments were carried out in pyruvate-free basal medium Eagle (BME). Where antioxidant protection against BD toxicity was tested, confluent cell monolayers were preincubated with DFO or 2-MAC for 6 h in Dulbecco's modified Eagle's medium. The antioxidant-containing medium was replaced by BME before BD treatment. Catalase was prepared in BME, and BD, 2-MAC, and DFO were prepared in deionized water. The stocks were prepared such that 10 μl of the stock was added to BME to provide the desired concentration for the incubation studies. BD and catalase were prepared immediately before use, while 2-MAC and DFO were prepared weekly and refrigerated between uses.
Lactate Dehydrogenase Measurement
Lactate dehydrogenase (LDH) release in the supernatant and cells was measured by the kinetic method of Wahlefeld as described previously (10). Then LDH release in the supernatant was calculated as percentage of total release, i.e., LDH release in supernatant + LDH release in cell fraction.
H2O2 accumulation in the supernatant was determined by incubating the supernatant with phenol red reagent for 30 min followed by addition of sodium hydroxide as described by Pick and Keisari (11). End-point color in H2O2 standards and samples was assayed with the spectrophotometer set at 610 nm. H2O2 levels were indexed for cell protein measured by the method of Lowry, and presented as μmol of H2O2 accumulated/mg protein per h.
Lipid Peroxidation Determination
Lipid peroxidation was assayed by quantifying malondialdehyde (MDA), one of its end products, in the form of thiobarbituric acid-reactive substances (TBARS) using the thiobarbituric acid reaction. TBARS were measured at 532 nm in the combined fractions of the supernatant and cell lysate and then corrected for protein using the Lowry method. To minimize lipid peroxidation during sample processing, 5 μl of antioxidant butylated hydroxytoluene (2%) was included while retrieving the samples.
The cell culture solutions and media were purchased from Life Technologies (Grand Island, NY), and culture flasks and plates were obtained from Costar (Acton, MA). The Bidrin® (formulation) was a gift from GemChem, Inc. (Ridgeland, MS), a distributor for Amvac Chemical Corp. (Los Angeles, CA). The BD formulation is made up of dicrotophos ((E)-2-dimethylcarbamoyl-1-methylvinyl dimethyl phosphate), which is the active ingredient (82%), and other ingredients (18%) some of which also have toxic potential such as isopropyl alcohol. BD is a brown liquid with a mild odor, stable at normal temperatures and completely miscible in water. The formula weight of dicrotophos is 237. Antioxidant 2-MAC was obtained as a gift from Upjohn Laboratories (Kalamazoo, MI). All other chemicals including DFO were purchased from Sigma Chemical Co. (St. Louis, MO) and were of analytical grade or the highest grade available.
BD-Induced Cell Injury: Effects of Concentration and Duration. Confluent LLC-PK1 cells were incubated with BD concentrations of 1000, 1250, 1500, 1750, and 2000 ppm for 6, 12, 24, and 48 h to determine concentration- and duration-dependent cell injury.
BD-Induced Cell Injury: Effects of 2-MAC and DFO. Graded concentrations of 2-MAC (0.3125, 0.625, 1.25 and 2.5 μM) and DFO (0.25, 0.5, 1 and 2 mM) were used to assess effects of these antioxidants on 1250 ppm BD-induced cell lysis after 24h of incubation. Cell damage was determined by LDH (% of total) release. These antioxidant protection results were used to select the 2-MAC and DFO concentrations for subsequent studies examining BD-induced H2O2 production and lipid peroxidation.
BD-Induced H2O2 Accumulation. Confluent cells were exposed to 1250 ppm of BD with or without preincubation with 2.5 μM 2-MAC or 2 mM DFO. Catalase enzyme (100 μg/ml) was coincubated with BD in some wells to verify H2O2 generation. BD treatment was given for 6 h. Then the medium was aspirated off and cells were incubated for another 12 h in BME alone. H2O2 accumulation in the supernatant was determined as a function of the total cell protein. The medium containing BD was removed to minimize interaction of BD with the reagents used in H2O2 measurement.
BD-Induced Lipid Peroxidation. Confluent cells were exposed to 1250 ppm BD with or without preincubation with a series of concentrations of 2-MAC (0.3125, 0.625, 1.25, and 2.5 μM) or DFO (0.25, 0.5, 1, and 2 mM). Cells were incubated with BD for 6 h. Then the medium was removed, and incubation was continued for another 12 h in BME, after which TBARS were measured. Medium containing BD was removed to minimize interference of any of this compound with the protocol used to assess TBARS.
Effect of H2O2 Scavenger Pyruvate on BD-Induced LDH Release and Lipid Peroxidation. Because pyruvate has been shown to scavenge H2O2 and protects LLC-PK1 cells against H2O2-induced cytolysis (12), cells subjected to 1250 ppm BD were also coincubated with 2 or 10 mM sodium pyruvate. The control cells without BD received equimolar sodium chloride. After 24 h of incubation, LDH release and MDA were measured.
Results are presented as mean ± SEM. Means were obtained from three experiments performed at least in duplicate. Statistical analysis was conducted using the StatView program from Abacus Concepts (Berkeley, CA). The differences between the groups were determined by ANOVA followed by Fisher (post hoc) test for comparison of multiple means. The level of significance was set at P < 0.05.
BD-Induced Cell Injury: Effects of Concentration and Duration
Exposure to BD resulted in a concentration- and time-dependent augmentation in cellular injury quantified by LDH leakage from cells (% of total LDH). At 6 h, all of the BD-treated groups, i.e., BD 1000 ppm (11.0 ± 0.58), BD 1250 ppm (13.67 ± 0.33), BD 1500 ppm (15.33 ± 0.33), BD 1750 ppm (16.67 ± 0.33) and BD 2000 ppm (16.67 ± 0.67), had significantly elevated levels of LDH (n = 3, ANOVA, P < 0.05, mean ± SEM) compared with the control (9.0 ± 0.58). Furthermore, BD 1000 ppm was significantly lower than all the other concentrations of BD, whereas BD 1250 ppm was significantly lower than all the other concentrations of BD except BD 1000 ppm. An acute increase in toxic response ranging from about two- to threefold over the control was observed at 12 h after incubation with 1000 to 2000 ppm of BD (Figure 1A). Cell injury was heightened with increase in duration of exposure. Continued incubation with BD for 24 h (Figure 1B) resulted in increased LDH release that was approximately fourfold above the control at the highest concentration, i.e., BD 2000 ppm. After 48 h of incubation, LDH release was significantly increased (n = 3, ANOVA, P < 0.05, mean ± SEM) in all BD-treated groups, i.e., BD 1000 ppm (40.33 ± 1.45), BD 1250 ppm (56.00 ± 4.16), BD 1500 ppm (83.67 ± 7.54), BD 1750 ppm (87.33 ± 1.45), and BD 2000 ppm (88.67 ± 2.03), compared with the control (25.33 ± 0.33). BD 1000 ppm was significantly lower than all the other concentrations of BD, whereas BD 1250 ppm was found to be significantly lower than BD 1500 ppm, BD 1750 ppm, and BD 2000 ppm. At 24 and 48 h, LDH release induced by 1250 ppm of BD was approximately equal to and about 2 times higher than the control (Figure 2). The 1250 ppm concentration of BD given over 24 h was thus selected for studying protection afforded by the antioxidants 2-MAC and DFO.
BD-Induced Cell Injury: Effects of 2-MAC and DFO
Preexposure of cells to 2-MAC (Figure 3) and DFO (Figure 4) resulted in a statistically significant suppression of LDH release (% of total) at 24 h, induced by 1250 ppm of BD. Maximum protection was afforded by 2.5 μM 2-MAC and 2 mM DFO, which amounted to a reduction in LDH leakage of about 21 and 18%, respectively, below that released by cells treated with BD alone. The 2-MAC and DFO concentrations used in this study did not have any effect on LDH release in cells incubated with BME alone (data not shown).
BD-Induced H2O2 Accumulation
BD-treated cells exhibited significantly higher catalase-inhibitable H2O2 accumulation (μmol/mg protein per h) compared with the control cells: 1.07 ± 0.023 versus 0.89 ± 0.029, P < 0.05 (Figure 5). The antioxidants 2-MAC and DFO were found not to influence H2O2 levels (data is not provided).
BD-Induced Lipid Peroxidation
Cells incubated with BD produced a significantly higher peroxidation of cellular lipids evidenced by MDA production (nmol/mg protein). Preloading of cells with 2-MAC (Figure 6A) and DFO (Figure 6B) decreased MDA levels concentration-dependently compared to cells treated with BD alone. After preexposure to 2.5 μM of 2-MAC and 2 mM DFO, a marked suppression of TBARS generation of approximately threefold was observed for both of the antioxidants, compared with the BD-treated group. More importantly, MDA formation in the group pretreated with 2.5 μM 2-MAC was significantly lower than the group given BD alone but was not significantly higher than the control.
Effect of H2O2 Scavenger Pyruvate on BD-Induced LDH Release and Lipid Peroxidation
Sodium pyruvate at 10 mM concentration suppressed 1250 ppm BD-induced LDH release and MDA formation, thus confirming the earlier results in this study that Bidrin, in part through the generation of H2O2 causes renal tubular cytoxicity and lipid peroxidation (Figure 7, A and B).
Our study shows that BD causes a dose- and time-dependent renal tubular cytotoxicity in association with H2O2 accumulation and lipid peroxidation, and that two chemically dissimilar antioxidants and a scavenger for H2O2 were able to reduce lipid peroxidation and prevent BD-induced cell injury.
The present study was undertaken in cell culture, using LLC-PK1 cells. They are an analog of proximal tubular cells and, although primarily anerobic, are well-characterized and have been used widely in toxin- and oxidant-related cell culture studies (6,7,8,9, 13). Although BD does not seem to pose any chronic toxicity in humans, acute intoxication has been associated with acute tubular necrosis (3). Our finding that BD induces H2O2 production and lipid peroxidation in a kidney cell line is consistent with the similar observations in OP-induced liver and neural toxicity (14,15,16,17). Others have provided additional evidence for the occurrence of OP-induced oxidative tissue damage evidenced by DNA-strand breaks (17), increased activities of antioxidant enzymes, and downregulation of glutathione peroxidase activity and glutathione (15, 16). Furthermore, studies have shown that antioxidants and cytochrome P-450 inhibitors suppress OP-induced MDA production and hepatic cytotoxicity (14). These reports in nonrenal tissues along with the observation of Berndt et al. that renal damage might be independent of AChE inhibition support our finding that BD through free radical mechanism may directly contribute to renal cell injury (5).
Two antioxidants with different chemical makeup were used to examine the involvement of ROS in BD-induced renal tubular cytoxicity. DFO inhibits iron-catalyzed peroxidation by chelating free iron, whereas 2-MAC, a hybrid of two potent antioxidants—α-tocopherol and 21-Aminosteroid—inhibits lipid peroxidation mainly by scavenging lipid radicals and thus limiting the lipid peroxidation chain reaction (18, 19). Our data indicate that although the antioxidants inhibited lipid peroxidation almost completely, cytoprotection was partial, suggesting the presence of other oxidant-induced injury pathways (Figure 8). Mechanisms other than lipid peroxidation include ATP depletion, DNA damage, protein oxidation, and intracellular calcium increase due to membrane permeability lesions. Additionally, processes not mediated by ROS may compromise cell viability by interacting with plasma membrane components, DNA, and proteins. Genotoxicity, inhibition of protein synthesis, and other cytotoxicity probably arising from processes such as alkylation of nucleic acids and/or alkylation and phosphorylation of proteins have been demonstrated in mammals after exposure to OP (20,21,22). Although lipid alterations such as degradation and peroxidation are major participants during early oxidant-induced LLC-PK1 cytotoxicity (10, 23), and certain antioxidants including 2-MAC afford protection against this early cytotoxicity, our present findings are in agreement with other reports (13) indicating that other mechanisms may come into play during later stages of oxidant-induced cytotoxicity.
Results from this investigation demonstrate that incubation of renal proximal tubular cells with BD is associated with H2O2 production, lipid peroxidation, and cell injury and that antioxidant suppressors of lipid peroxidation and free radicals protect against this form of cell injury. Based on this finding, we hypothesize that ROS generated during BD intoxication may be responsible for at least some of the morphologic and pathophysiologic changes observed in human and rat kidneys (3, 5, 24).
The source or the mechanisms for increased levels of H2O2 in BD-treated cells is not explored in this work. Additional studies are needed to determine whether BD-induced activation of NAD(P)H oxidase system, increased mitochondrial activity, reduced antioxidant activity, or a combination of these would account for the increased ROS levels. It is important to note that although cell culture systems are convenient and useful for monitoring many cellular events, the concentrations of BD used in this setting may not reflect the varying in vivo tubular cell concentrations, and that the BD effect is independent of any hemodynamic effects invariable in the in vivo setting. Therefore, in vivo studies and studies in humans exposed to BD or other OP are necessary to test our hypothesis that free radicals play a role in acute tubular necrosis after the high dose OP intoxication.
This work was supported in part by the Title III Graduate Research Assistantship awarded through Jackson State University (Jackson, MS) to Dr. Poovala, and by a Baxter extramural grant and National Institute of Environmental Health Sciences Grant RO3-ES 09264-01 to Dr. Salahudeen. We thank Dr. Abdul Mohamed, Dr. Hiroyasu Tachikawa, and Dr. Paul Tchounwou of Jackson State University, and Dr. John Bower of the University of Mississippi Medical Center for their continued support. We also thank GemChem, Inc., and Amvac Chemical Corp. for the generous gift of Bidrin®, and Upjohn Laboratories for donating 2-methylaminochroman.
American Society of Nephrology
This work was presented as an abstract at the Mississippi Academy of Sciences Annual Meeting held in February 1998 in Biloxi, Mississippi.
aBidrin® is a registered trademark of Amvac Chemical Corp. (Los Angeles, CA).
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