Dig1 protects against cell death provoked by glyphosate-based herbicides in human liver cell lines
© Gasnier et al; licensee BioMed Central Ltd. 2010
Received: 15 June 2010
Accepted: 27 October 2010
Published: 27 October 2010
Worldwide used pesticides containing different adjuvants like Roundup formulations, which are glyphosate-based herbicides, can provoke some in vivo toxicity and in human cells. These pesticides are commonly found in the environment, surface waters and as food residues of Roundup tolerant genetically modified plants. In order to know their effects on cells from liver, a major detoxification organ, we have studied their mechanism of action and possible protection by precise medicinal plant extracts called Dig1.
The cytotoxicity pathways of four formulations of glyphosate-based herbicides were studied using human hepatic cell lines HepG2 and Hep3B, known models to study xenobiotic effects. We monitored mitochondrial succinate dehydrogenase activity and caspases 3/7 for cell mortality and protection by Dig1, as well as cytochromes P450 1A1, 1A2, 3A4 and 2C9 and glutathione-S-transferase to approach the mechanism of actions.
All the four Roundup formulations provoke liver cell death, with adjuvants having stronger effects than glyphosate alone. Hep3B are 3-5 times more sensitive over 48 h. Caspases 3/7 are greatly activated in HepG2 by Roundup at non-cytotoxic levels, and some apoptosis induction by Roundup is possible together with necrosis. CYP3A4 is specifically enhanced by Roundup at doses 400 times less than used in agriculture (2%). CYP1A2 is increased to a lesser extent together with glutathione-S-transferase (GST) down-regulation. Dig 1, non cytotoxic and not inducing caspases by itself, is able to prevent Roundup-induced cell death in a time-dependant manner with an important efficiency of up to 89%, within 48 h. In addition, we evidenced that it prevents Caspases 3/7 activation and CYP3A4 enhancement, and not GST reduction, but in turn it slightly inhibited CYP2C9 when added before Roundup.
Roundup is able to provoke intracellular disruption in hepatic cell lines at different levels, but a mixture of medicinal plant extracts Dig1 can protect to some extent human cell lines against this pollutants. All this system constitutes a tool for studying liver intoxication and detoxification.
Roundup (R) is the most widely used non-selective herbicide worldwide. It is comprised of a mixture of an isopropylamine salt of glyphosate (G) and adjuvants. G is considered as the active ingredient of R, although quantitatively it is a minor constituent, which is not supposed to be toxic alone in mammals . Various adjuvants are present in R as secret of formulations , amplifying and thus allowing the G herbicide action, as well as its unintended toxic and endocrine disrupting effects in human placental cells . The adjuvants, which are chosen from a long list that can vary from formulation to formulation , stabilize and help G penetration into cells. Among these are benzisothiazolone, isobutene, light aromatic petroleum distillate, methyl pyrrolidinone, polyethoxylated tallowamine or alkylamine (POEA), etc . Some of these compounds may be genotoxic or form adducts with DNA . It is thus important to compare different R formulations when studying this herbicide's toxicity. Moreover R residues are quite stable in rivers and soils . G and its metabolite AMPA (aminomethyl phosphonic acid) are among the primary pollutants of surface waters (IFEN, 2006); they also enter the food chain . These chemicals are found in the urine of agricultural workers . The use of this herbicide is increasing as more than 75% of genetically modified edible plants have been designed to be used in conjunction with R. These plants are engineered to tolerate high intracellular levels of R . We have also shown that the human embryonic kidney 293 cell line was even more sensitive to R, this was dose- and time-dependent ; and thus it was hypothesized that this could explain pregnancy outcomes and miscarriages reported for agricultural workers using G-based herbicides . This is consistent with the fact that G-based herbicides have recently been shown to be endocrine disruptors in cell lines .
We know that xenobiotics have a main endpoint in the liver, which is the major detoxification organ. Here, we investigated the mechanism of action of R in the human liver cell lines available, HepG2 and Hep3B, which have been used as a model system to study xenobiotic toxicity, most prominently HepG2 [11, 12]. We wanted to compare in the first instance the actions of four R formulations on both cell lines and then to detail the enzymatic pathways activated in HepG2.
Detoxifying mechanisms are frequently enhanced by plant extracts, which can provide additional protection against radicals and electrophilic compounds [13, 14]. We have tested the ability of a new drug described for the first time, Dig1 (D), to protect cells from R intoxication. D contains plant extracts from Taraxacum officinalis, Arctium lappa and Berberis vulgaris. These herbal preparations were chosen in particular for their digestive detoxification or hepato-protective effects [15–20]. It was thus interesting to compare these general findings on plant extracts to some biochemically precise markers that could be modified in human hepatocytes, such as caspases 3/7, cytochromes P450, glutathione S-transferase (GST), and mitochondrial succinate dehydrogenase (SD), in order to detail the pathway(s) of action(s) of these mixtures used as medicinal plants in vivo, and thus to explore their cellular protective potential.
Four main R formulations which have been used in agriculture (Monsanto, Anvers, Belgium) have been chosen in this study: Express® 7.2 g/l of G called glyphosate or N-(phosphonomethyl) glycine, product number 2010321; Bioforce® 360 g/l of G, product number 9800036; GT® 400 g/l of G, product number 8800425; GT+® 450 g/l of G, product number 2020448. The various herbicide formulations were prepared in Eagle's modified minimum essential medium (EMEM; Abcys, Paris, France), with 10% calf fetal serum from Cambrex (Verviers, Belgium) otherwise specified. G was from Sigma-Aldrich (Saint Quentin Fallavier, France), its called "2% solution" was equivalent in concentration to 2% R Bioforce® and was prepared in serum free-medium, and adjusted to pH 5.8 of 2% R. D is a mixture of diluted plant extracts obtained by Sevene Pharma (Monoblet, France) from original independent macerates corresponding to 1/10 of dried plants in a water-alcohool solution of 45 to 55%. They are afterwards diluted in 70% alcohol with Taraxacum officinalis macerate at 10-4, Arctium lappa at 10-4 and Berberis vulgaris at 10-5. D is prepared in the medium at 2% of the mixture in positive controls. The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and all other compounds, unless otherwise specified, were from Sigma-Aldrich. The MTT stock solution at 5 mg/ml in phosphate-buffered saline was diluted 10-fold in serum-free EMEM and then filtered through a 0.22 μm filter.
2. Cell cultures, Roundup and/or Dig1 exposures
3. S9 fractions
The medium was removed, and cells dislodged by treatment with 7 ml of trypsin-EDTA (Lonza, France) and washed (PBS, Eurobio, France) twice by centrifugations (70 g, 5 min), at room temperature. Cells were then resuspended in 500 μl of 50 mM phosphate buffer pH 7.5 with 0.25 M sucrose, 1 mM DTT, homogenized and centrifuged at 9,000 g, 4°C for 30 min. The supernatants corresponding to the S9 fractions (membrane and cytosolic enzymes) were collected and frozen at -80°C until further evaluation for enzyme activities. Protein concentration was determined in each S9 fraction according to the Bicinchoninic Acid Protein Assay (Sigma, France).
4. Cell death measurement
The enzymatic MTT test is based on the cleavage of MTT into blue formazan by the mitochondrial enzyme succinate-dehydrogenase [21, 22], it was used to evaluate human cell viability as described in our group . The optical density was measured using a luminometer (Mithras LB 940, Berthold, France) at 570 nm. The crude protective actions were evaluated at the end of the treatment, by comparing the toxicity of R after treatment by D or not. As R toxicity is induced at the chosen LC50, the relative efficiency of the protective effect (recovering) is the percentage of recovered viability in the presence of D in comparison to the maximal toxic effect at LC50.
5. Caspase 3/7 activity measurement
The Caspase-Glo® 3/7 assay (Promega, Paris, France) in 96-well white plates (Dutscher, France) was a luminescent method designed for automated high-throughput screening of caspases activity, which is a measure of apoptosis. This method can measure caspase-3 and -7 activities in purified enzyme preparations or cultures of adherent or suspended cells [24–26]. The assay provides a pro-luminescent caspase-3/7 substrate, which contains the tetrapeptide sequence DEVD. This substrate is cleaved to release amino-luciferin, a substrate for luciferase, and the production of light is proportional to the quantity of amino-luciferin released and therefore proportional to caspase. The Caspase-Glo® 3/7 reagent has been optimized for caspase activity, luciferase activity and cell lysis. The addition of the single Caspase-Glo® 3/7 reagent, in an "add-mix-measure" format, results in cell lysis followed by caspase cleavage of the substrate and generation of a "glow-type" luminescent signal. After cell cultures were exposed to 50 μL of various dilutions, an equal volume of Caspase-Glo® 3/7 reagent was added to each well. Plates were then agitated 15 min and incubated 45 min at room temperature in the dark, to stabilize the signal before measuring luminescence. The negative control was the serum-free medium, the positive control was the active Caspase-Glo® 3/7 reagent mixed with cells treated only with serum-free medium to determine the basal activity of the caspases 3/7. Luminescence was measured using the luminometer Mithras LB 940 (Berthold, Thoiry, France) at 565 nm.
6. Cytochrome P450 activity measurement
The cytochrome P450 3A4, 2C9, 1A2 and 1A1 activities were quantified by the P450 Glo™ Assays (Promega, France), as described by Yueh et al. . Each Cytochrome P450/1 M KPO4/Substrate Reaction mixture containing the S9 fractions (duplicate) was pre-incubated at 37°C for 10 min in white 96-well plates (Dutscher, France). The Cytochrome P450 CYP1A1 Reaction mixture contained 135 μg of the human liver S9 fraction as control (Tebu-Bio, France) or cell S9 fraction with 60 μM luciferin-conjugated substrate (luciferin-6'-chloroethyl ether) and 200 mM KPO4 buffer in a final volume of 25 μl. For CYP1A2 130 μg S9 fractions were used with 200 μM substrate of luciferin-6'-methylether; for CYP2C9 160 μg S9 and 200 μM substrate of 6'-deoxy luciferin but with 50 mM KPO4 buffer in 25 μl. CYP3A4 was measured with 170 μg S9 with 100 μM substrate of luciferin-6'-benzylether but with 400 mM KPO4 in 25 μl. The enzymatic reaction was initiated by adding 25 μl of NADPH regenerating system to each well. It contained 2.6 mM NADP+, 6.6 mM glucose-6-phosphate, 0.8 U/ml glucose-6-phosphate dehydrogenase and 6.6 mM MgCl2.
The plate was then incubated at 37°C for 20 min for CYP1A1 and CYP1A2, and, for 30 min for CYP2C9 and CYP3A4. The reconstituted Luciferin Detection reagent (50 μl) was added before mixing for 10 s and incubating at room temperature for 90 min in order to stabilize the luminescent signal. The luminescence was then read with a luminometer (Veritas Turner Biosystems). Three independent experiments were carried out using three independent batches of S9 fractions.
7. GST activity measurement
The protocol was adapted from Habig et al. . Briefly, 320 μg (50 μl) of the human liver S9 fraction (positive control) or cell S9 fraction was mixed with 10 μl of 100 mM GSH and 930 μl phosphate buffer in duplicate. Reduced L-glutathione (GSH) was dissolved in deionized water; pH 6.5 buffer was prepared by mixing 0.7 volume of 0.1 M KH2PO4 and 0.3 volume of 0.1 M Na2HPO4. The reaction was initiated by 10 μl of 100 mM 1-chloro-2,4-dinitrobenzene (CDNB) substrate. The CDNB was dissolved in 95% ethanol at a concentration of 100 mM (20.3 mg/ml). After a 90 s incubation at 37°C, the optical density was measured at 340 nm every 30 s for 90 s with a SmartSpec 3000 Spectrophotometer (Bio-Rad, France). Three independent experiments were carried out using three independent batches of S9 fraction.
8. Statistical analysis
The experiments were repeated 3 times in different weeks in triplicate (n = 9) unless otherwise specified. All data are presented as the mean ± standard error (S.E.M.). Statistical differences were determined by a Student unpaired t-test using significant levels of p < 0.01 (**) and p < 0.05 (*).
This work evidences for the first time the effects of several formulations of the major herbicide worldwide (R) on human hepatic cell lines available, which are widely recognized as models to study xenobiotic actions. We tested R at sub-agricultural levels; the LC50 measured in this work is 10 times for Hep3B and 4 times for HepG2 below the maximum level of residues authorized in some feed (400 ppm, ). We found that the cell sensitivity depended on the nature of the R formulation. Both cell lines have retained the activities of the drug metabolism phase I and phase II enzymes involved in activation and detoxification of genotoxic carcinogens [30, 31]. HepG2 cells are in general considered a better model since they have three-fold higher levels of CYP1A1 and glutathione-S-transferase (GST) than Hep3B . For instance, it was observed that HepG2 are more sensitive than Hep3B to cisplatin , to dietary genotoxins  and to genotoxicants . In our hands it was the opposite for R, overall in 48 h. In fact, both cell lines are from different genetic origins, from different boys at different ages, and thus have specific enzymatic equipments including cytochromes P450. Greater sensitivity was observed with Hep3B. It was thus important to obtain results in both lines that confirmed R toxicity in all human models tested up to now, including embryonic 293 cells, umbilical cord cells, placental derived JEG3 cells and microsomes from fresh placenta [3, 4, 34]. It was evidenced that cell death increased with time of exposure for all R formulations, this phenomenon shows that the threshold of toxicity depends not only on the dose but also on time, as previously clearly demonstrated on embryonic and placental cells . It was also shown that toxicity was dependent on the nature of the adjuvants present in different R formulations. Moreover, the action of serum only temporarily buffered R toxicity. We found that serum-free culture medium revealed essentially the same xenobiotic impacts that are visible 1-2 days later in serum. Thus, although the timecourse of action is delayed, the pathways of actions appear similar .
Indeed, the G cytotoxic effects do not vary linearly with dose; this demonstrates the differential roles of adjuvants in the amplification of toxicity, since G has no toxicity alone at these concentrations. The adjuvants added to G in various R formulations are considered manufacturing secrets, but obviously do not form an inert part of the composition. One mechanism of adjuvant action is most probably to form detergent vesicles that allow cell membrane opening and penetration of G, and that most probably facilitate bioaccumulation of G, metabolites and adjuvants, and gene disruptive effects, which could explain time-amplified effects. A very small quantity of adjuvants combined with G has been already demonstrated to have similar effects to R . It is a recognized fact that mixtures of xenobiotics have synergistic effects . We observed that whatever the nature of the various adjuvants is in the 4 R formulations, the mechanism of toxicity is similar on several crucial endpoints: namely SD, AK, Caspases 3/7 . Only the threshold of toxicity is different.
In this study, we sought to understand the mechanism of toxicity of the two R formulations that have the most rapid toxic effects on hepatic cells. We also evaluated whether it was possible to prevent R toxicity by D. D is a newly described product comprised of a mixture of extracts from Taraxacum officinalis, Arctium lappa and Berberis vulgaris. Taraxacum was cited for protective effects in the digestive system [19, 20], also anti-tumoral  and anti-oxydant effects . Arctium lappa is also found to be hepato-protective [17, 18], as well as Berberis .
First of all, the lack of D impact on cell viability in comparison to controls indicates that it is not cytotoxic at 2%. It was hypothesized first that either D does not penetrate cells without embedding cell-cell interactions, or it is relatively inert on several important markers of cell function at this concentration. Our results are consistent with D penetrating the cells and not just forming a shield that prevents R from penetrating. This is verified since effective protection by D necessitates more than 6 h of contact, and because D modifies enzymatic activities, as detailed below. As a matter of fact, D shows a clear and very strong efficiency of protective action against the toxicity of R in both hepatic cell lines; protection is up to 89% for HepG2 in 48 h. It is not completely excluded that this important action of D can occur by preventing entrance of R into cells, normally helped by adjuvants. However D clearly has intracellular actions, including prevention of caspases 3/7 activation and CYP3A4 enhancement, both provoked by R. CYP 3A4 was induced by Roundup in comparative manner and in similar time than with other xenobiotics Bisphenol A, DDT and phtalates, respectively [38–40].
Other metabolic/biochemical effects of D components have also been reported, for instance, the protective effects on acute pancreatitis in rats  through IL-6 reduction, as well as the suppression of reactive oxygen species, nitric oxide and lipid oxidation . Polyphenolic compounds present in D are considered hepatoprotective [42–44] but the time-dependent protective effects on the crucial mitochondrial succinate dehydrogenase and caspases 3 and 7 had not been demonstrated before.
Since HepG2 are more resistant and D more preventive on this line, we decided to detail the signalling pathway of R and D actions in these cells. Moreover, the caspases, cytochromes and GST activities are more fully documented in this hepatic cell line [45–47]. The measurement of caspase 3/7 activity has been recognized as a marker for apoptosis in HepG2 cells .
Caspases 3/7 have been rarely measured in HepG2, and found to be induced 200% by 30 μM Tamoxifen in 24 h . Their induction by R in our study (156% in 24 h and 765% in 48 h) confirms the proposed apoptotic mechanism activated by R. Of course, this does not exclude some necrosis as a primary mode of cell death as we previously showed . Our results also show that D alone does not interfere with caspases but may possibly prevent R impact on these enzymes.
At lower levels and even with the buffering effect of serum already documented , D also prevents CYP3A4 induction: CYP3A4 is by far the major target of R among the phase I activating enzymes measured. CYP3A4 is the most abundant P450 expressed (60%) in human liver . It is involved in the bioactivation of environmental procarcinogens, such as aflatoxin B1  and benzo[a]pyrene among a number of others . Some organochlorine pesticides can activate the PXR, a nuclear hormone receptor  that regulates CYP3A4 gene transcription in particular in liver. R components may also induce this process. The results with CYP1A2 (slight induction by R) show that its action does not depend on a unique pathway, like the results on CYP2C9. Only a few reports are available on CYP2C9, which is inhibited by extracts of other plants such as pineapple .
Similarly, only some xenobiotics such as polychlorobyphenyls inhibit GST; this is typical of estrogen-like effects  already observed with R on aromatase [3, 4]. The disruption of xenobiotic metabolism by R in our study, which increased some cytochromes P450 and decreased GST, may alter liver detoxification function, leading to the accumulation of toxic reactive oxydated molecules  in a manner that could be prevented by D in this study. In fact, GST inhibition, if R intoxication continues, could lead to the accumulation of reactive compounds, due to disruption of xenobiotic excretion.
In conclusion, it is demonstrated and explained that high hepatic cell line mortality is provoked by R, at doses far below those used in agriculture. The mechanism of action on various essential enzymes is detailed in Figure 8. This impact on cell death was observed at doses far smaller than legally allowed residues of G in GM food or feed (400 ppm, ), in our study LC50 was in comparison 40 to 96 ppm. Of course G can be metabolized and excreted out of the body but this has to be balanced in regard to its cell penetration and bioaccumulation due to adjuvants. In these conditions, this can be almost totally prevented in HepG2 in 48 h by a specific drug, D, that mostly prevents caspases 3/7 activation and CYP3A4 enhancement, both provoked by R. However it does not prevent GST inhibition by R that could lead to accumulation of toxic reactive compounds. Since the use of cell lines allows longer experiments, and since cell lines may in some instances be less sensitive to xenobiotics than primary cultures , this system with R and Dig1 constitutes an interesting tool for studying liver intoxication and detoxification. The activity of Dig1 should be now also tested in vivo in animal experiments.
This study was supported by Sevene Pharma Company which provided Dig 1. C.G., N.B., E.C. held fellowships from the Conseil Regional de Basse-Normandie and the CRIIGEN (Committee for Independent Research and Information on Genetic Engineering). C.G. fellowship was also supported by the Ethic Committee of Léa Nature Group/Jardin Bio which is gratefully acknowledged here. Part of the work was accomplished in C.RIS Pharma Company (Cytochromes and GST study) and in IMOGERE (University of Caen) for experiments with radioactive compounds. We would like also to thank the Human Earth Foundation and the Denis Guichard Foundation for structural support. We thank John Fagan for the English revision of the manuscript.
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