- Email: [email protected]
2 by insecticides, in hepatic or epidermal cells: binding capability to the Ah receptor
Toxicology Letters 96,97 (1998) 33 – 39
Cytotoxic effects and induction of cytochromes P450 1A1/2 by insecticides, in hepatic or epidermal cells: binding capability to the Ah receptor C. Delescluse a,*, N. Ledirac a, G. de Sousa a, M. Pralavorio a, P. Lesca b, R. Rahmani a a
Laboratoire de Pharmaco-toxicologie Cellulaire et Mole´culaire, Centre de Recherches INRA, 41 Bd du Cap, 06606 Antibes, France b Laboratoire de Pharmaco-toxicologie, Centre INRA, B.P. 3, 31931 Toulouse, France
Abstract Insecticides deserve particular attention since the general population is potentially exposed to such chemicals through many routes. We therefore tested the comparative acute and chronic toxicity of chemicals belonging to the major insecticides families (DDT, malathion and tetrachlorvinphos, carbaryl, cypermethrin, diflubenzuron), in hepatocytes, HepG2 and HaCaT cell lines. Two kinds of end-points were used: cytotoxicity parameters and CYP1A1 induction. Except for cypermethrin and diflubenzuron, all these chemicals exerted a cytotoxic effect in hepatocytes and HaCaT, but not in HepG2 cells. However, the induction of the EROD activity appeared more sensitive since a response was detected at lower concentrations. Significant differences were observed between the cell types and the insecticides. Furthermore, these chemicals were unable to displace [3H]TCDD from its binding sites, suggesting that they would not be a ligand of the Ah receptor. The experimental approach used herein may be a good means for predicting the acute and chronic toxicity of pesticides. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Insecticides; CYP1A1 induction; Cytotoxicity; Ah receptor
Abbre6iations: BSA, bovine serum albumin; DMEM, Dulbecco modified Eagle’s Medium; CBR, carbaryl; CPR, cypermethrin; CYP1A1, cytochrome P4501A1; DFU, diflubenzuron; EROD, ethoxyresorufin-O-deethylase; FCS, fetal calf serum; MAL, malathion; 3-MC, 3-methylcholanthrene; NADP, nicotinamide adenine dinucleotide phosphate; PBS, phosphate buffer solution; TCDD, 2,3,7,8-tetrachlorodibenzop-dioxin; TCDF, tetrachlorodibenzofuran; TCV, tetrachlorvinphos. * Corresponding author.
1. Introduction Pesticides represent high volume and widely used environment chemicals. Annual commercial production averages 5×108 kg in the USA, with 1800 chemicals registered so far into 22000 different formulations and a retail sales value of several billions dollars. They contrast with other chemical classes such as occupational hazards or
0378-4274/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0378-4274(98)00047-2
34
C. Delescluse et al. / Toxicology Letters 96,97 (1998) 33–39
therapeutic drugs, in that they necessarily are toxic to selected biological systems and are deliberately deposited into the environment. Moreover, due to their lipophilic nature and slow chemical and biological degradation, these molecules tend to be taken up by biological membranes and tissues, concentrated in organisms, and progress up the food chain (Safe, 1986; Ahlborg et al., 1992; Dubois et al., 1996). This bioaccumulation and the resultant constant exposure either via direct exposure or food consumption and drinking water may cause a gradual deterioration of health. There is therefore a continuous debate concerning their possible role in various chronic effects in human, including neurotoxicity, development and reproductive effects, cyto- and genotoxicity, enzyme induction… (Kurtz et al., 1989; Chambers and Carr, 1995). In that context, we aimed at assessing the acute and chronic toxicity of various compounds belonging to the major insecticides families: DDT for chlorinated hydrocarbons, malathion (MAL) and tetrachlorvinphos (TCV) for organophosphorus, carbaryl (CBR) for carbamates, cypermethrin (CPR) for pyrethroids and diflubenzuron (DFU) for benzoylurea. As the main potential portals of entry of insecticides are the skin (in case of dermal contact) and the liver (in case of ingestion), two kinds of cellular models were used: (i) epidermal cells such as spontaneously immortalised keratinocytes HaCat cell line (Boukamp et al., 1988); (ii) hepatic cells such as human and rat hepatocytes, as well as the HepG2 human hepatoma cell line. These models were also selected because they possess the enzyme equipment to bioactivate or detoxify xenobiotics (Delescluse et al., 1997). As concerns toxicological end-points, different kinds of parameters were used: (i) cytotoxicity parameters, namely MTT and neutral red tests for mitochondrial and lysosomal functions respectively; (ii) parameters measuring the chronic effects of xenobiotics, such as the induction of cytochrome P450 1A1/2 expression which is considered to be one of the most sensitive and specific cellular response to harmful xenobiotics. The current study highlight the interest of using various cell types and end-points for evaluating or predicting the potential acute and chronic toxicity of environmental pollutants.
Table 1 Comparative in vitro cytotoxicity of insecticides determined by the MTT test, after 48 h exposure I.C.50 (mM)
DDT MAL TCV CBR CPR DFU
Hepatocytes
HepG2
HaCaT
250 253 40 264 \1000 \1000
\100 \100 \100 \100 \100 \100
70 20 77 \100 \100 \100
Results are representative of experiments using HaCaT cells and human hepatocytes (mean of five replicate). Cells were treated for 48 h with various concentrations of pesticides and the MTT test was performed as described in Section 2. Pesticide solutions were prepared in DMSO, and the final concentration of DMSO never exceeded 0.5% (v/v).
2. Materials and methods
2.1. Chemicals Dulbecco modified Eagle’s medium (DMEM), penicillin–streptomycin, L-glutamine, sodium pyruvate, Eagle’s non-essential amino-acids, fetal calf serum (FCS), were from Bioproducts (Germany). William’s medium, DMSO, glucose-6-
Fig. 1. In vitro cytotoxicity of insecticides as determined by the MTT test, after 48 h of exposure. Results are representative of experiments using human hepatocytes ( — ) and spontaneously immortalized keratinocytes, HaCaT cells ( – – – ). Each point is the mean of at least five replicates.
C. Delescluse et al. / Toxicology Letters 96,97 (1998) 33–39
35
Fig. 2. Maximal inducing effect of the different insecticides on rat hepatocytes (HEP), HepG2 and HaCaT cells, by reference to 3-MC. EROD activity was determined as described in Section 2. Results are expressed as the means 9S.D. for at least two independent experiments in triplicate. Chemical concentrations: 3-MC (0.5 mM), MAL (50 mM), DDT (10 mM), CBR (100 mM), CPR (100 mM), DFU (100 mM), TCV (75 mM). Asterisks indicate significant differences from control (*, P B0.05; **, P B0.001; one-way ANOVA and Newman–Keul’s post hoc test).
phosphate, glucose-6-phosphate dehydrogenase, b-NADP, 3-methycholanthrene (3-MC) were from Sigma (France). 7-ethoxyresorufin and resorufin were from Boehringer (Germany). Insecticides were from Cluzeau Info Labo (France). 2,3,7,8-tetrachlorodibenzofuran (TCDF) was from Chemsyn Sciences Lab (USA). Hydrocortisone hemisuccinate was from Roussel-Uclaf.
2.2. Cell culture and treatment Human hepatocytes were obtained from liver biopsies resected from secondary tumors, by the classical two-step collagenase perfusion technique (Fabre et al., 1988). Rat hepatocytes were isolated, as previously described (Berry and Friend, 1969), from male Sprague – Dawley weighing 220 – 240 g. Freshly isolated cells were resuspended in William’s medium containing 10% FCS and supplemented with penicillin (50 U/ml), streptomycin (50 mg/ml), and insulin (0.1 U/ml). Hepatocytes were seeded in collagen type I-coated dishes. Plates containing hepatocytes were incubated 4 h at 37°C under a humidified 5% CO2 atmosphere. The medium was then renewed with the same
initial medium without FCS but supplemented with hydrocortisone hemisuccinate (1mM), and containing increasing concentrations of the different pesticides. HepG2 and HaCaT were cultured in DMEM supplemented with 10% FCS, penicillin (100 U/ ml), streptomycin (100 mg/ml), sodium pyruvate (1 mM), non-essential amino acids (0.1 mM) and L-glutamine (2 mM). Cell cultures were subcultured every 4 days for HaCaT and 6 days for HepG2 at 1:10 and 1:2 ratios, respectively. Cells were seeded on 100 mm diameter plates for Northern blot analysis, or 96-well microtiter plates for EROD. Then, insecticides or 3-MC, dissolved in DMSO (final concentration 0.5%) were added to the cultures for 72 h, with a change of medium every 24 h.
2.3. Cytotoxicity test The cytotoxic effects of the insecticides were assessed after 48 or 72 hr of exposure, by using the MTT test for hepatocytes and cell lines, respectively. The assays were carried out as previously described (Fautrel et al., 1991).
36
C. Delescluse et al. / Toxicology Letters 96,97 (1998) 33–39
2.4. EROD (7 -ethoxyresorufin-O-deethylase) acti6ity assay The determination of the EROD enzyme activities in cells cultured for 3 days with the test compounds, was performed according to Reiners et al. (1990), Donato et al. (1993), with slight modifications. Briefly, after treatment, the medium was discarded and 25 ml of TMB buffer (Tris –HCl 100 mM pH 7.8, MgCl2 2.5 mM, BSA 0.06%) containing 1% glycerol was added per well and the cultures were frozen at −80°C. After thawing, 200 ml/well of TMB buffer containing glucose-6-phosphate (3 mM), NADP (0.5 mM), dicoumarol (10 mM), ethoxyresorufin (2 mM) and glucose-6-phosphate dehydrogenase (0.1 U/ml) were added. The EROD activity was measured at 37°C by spectrofluorimetry (lex =600 nm, lem = 535 nm) by following the kinetics of appearance of resorufin from ethoxyresorufin.
2.5. mRNA analysis Three days after treament, total RNA was isolated from the cell culture by the acidic phenol extraction procedure (Chomczynski and Shacchi, 1987). Twenty micrograms of RNA were sizefractionated on a 0.9% agarose gel containing 10% formaldehyde and transferred to a nitro-cellulose membrane. Hybridization was performed with 476 pb cDNA insert of human CYP1A1 mRNA corresponding to nucleotides +311 to +787 and labelled using 32P.
2.6. Ligand binding Three hundred microliters of enriched 9S fraction containing human Ah receptor were incubated with 40 nM of [3H]TCDD (2,3,7,8tetrachlorodibenzo-p-dioxin) in absence or presence of either 40 mM TCDF or 80 mM insecticides. [3H]TCDD, TCDF, (tetrachlorodibenzofuranin) and insecticides were dissolved in DMSO. Then, cytosol samples were analysed by velocity sedimentation in sucrose gradient (10– 30%). Gradients were centrifuged at 4°C for 2 h (372000 g), and 22 fractions (282 ml; eight drops per fraction) were collected. The radioactivity of each fraction was determined by liquid scintillation counting (Lesca et al., 1987).
2.7. Statistical analysis
Fig. 3. Induction of CYP1A1/2 mRNA in cultures of human hepatocytes, HepG2 cells and HaCaT cells. Cells were treated either with DMSO or various concentrations of chemicals for 72 h, with a change of medium every day. 10 mg (hepatocytes) or 20– 30 mg (HepG2 and HaCaT) of total RNA was analysed and CYP1A1/2 mRNAs were revealed with a radiolabeled CYP1A1/2 probe as described in Section 2. Chemical concentrations: (lane 1) DMSO 0.5%; (lane 2) 3-MC 2 mM (hepatocytes) and 1 mM (HepG2 and HaCaT cells); (lane 3) DFU 50 mM; (lane 4) TCV 25 mM (hepatocytes) and 50 mM (HepG2 and HaCaT cells); (lanes 5) DDT 10 mM (hepatocytes) and 50 mM (HepG2 and HaCaT cells); (lane 6) CBR 75 mM.
All enzymatic results were evaluated using a one-way analysis of variance for control and insecticide-treated cells. The statistical significance was calculated using Newman–Keuls post hoc test for multiple mean comparisons against the control set. Cytoxicity data were standardized as the percentage of control cell viability.
3. Results and Discussion
3.1. Cytotoxicity Cell viability was estimated by the MTT test. As shown in Table 1 which reports the comparative cytotoxicity (IC50 values) for the various in-
C. Delescluse et al. / Toxicology Letters 96,97 (1998) 33–39
37
Fig. 4. Absence of competitive binding of various insecticides on the Ah receptor. The enriched fraction from human hepatocyte cytosols were incubated with 40 nM [3H]TCDD, in absence or presence of 80 mM insecticides or 40 mM TCDF, for 2 h at 4°C. Then samples were analysed by velocity sedimentation on 10–30% sucrose gradients as described in Section 2.
secticides on different cell types, significant differences were observed between the tested compounds. Except cypermethrin and diflubenzuron, all these chemicals exerted a cytotoxic effect with a IC50 ranging from 20 to 264 mM in hepatocytes and HaCaT, but not in HepG2 cells. Tetrachlorvinphos was the most toxic derivative for human hepatocytes whereas it was malathion in
HaCaT cell line. However, it should be pointed out that due to the poor solubility of some of the insecticides in culture medium, IC50s may be under-estimated. Fig. 1 illustrates the dose-dependent toxic effect of the three most toxic insecticides and confirms that hepatocytes and HaCaT have different sensitivity toward these compounds.
38
C. Delescluse et al. / Toxicology Letters 96,97 (1998) 33–39
3.2. CYP1A1 induction The ability of the six insecticides in inducing EROD activity was tested in human and rat hepatocytes, HepG2 and HaCaT cell lines, at various non cytotoxic concentrations. Fig. 2 shows the maximal inducing effect of the insecticides in rat cells, by reference to the positive control 3-MC. This test appeared much more sensitive than cytotoxic tests, since a response to insecticides was detected at concentrations as low as 10 mM. Moreover, significant differences were observed between the cell types. Four compounds, namely carbaryl, cypermethrin, diflubenzuron and tetrachlorvinphos (Fig. 2) were shown to significantly activate the EROD activity: from 6 to 9-fold-over control DMSO treated cells in hepatocytes. The effect of the strongest inducers: diflubenzuron and tetrachlorvinphos was dose-dependent (de Sousa et al., 1997). These results were confirmed by northern blot analysis for diflubenzuron, but surprisingly not for tetrachlorvinphos (Fig. 3). This observation could be explained by a more rapid degradation of mRNAs following tetrachlorvinphos treatment, or by different regulation mechanisms for CYP1A1/2 gene expression by the two compounds. Different observations were done in HaCaT and HepG2 cells, where only carbaryl was able to significantly induce the EROD activity (Fig. 2). This effect is dosedependent in both cell types (Ledirac et al., 1997) and is confirmed by northern blot analysis of CYP1A1 mRNAs (Fig. 3). On the other hand, diflubenzuron and tetrachlorvinphos inhibit basal EROD activity (Figs. 2 and 3) in these cell lines whereas they were inducers in hepatocytes.
3.3. Binding capability to the Ah receptor To further confirm these results, we examined the capability of these chemicals to bind the human hepatic Ah receptor (AhR). Competitive binding assays were performed by incubating the 9S enriched fraction of human liver cytosol with [3H]TCDD at 40 nM, in absence or presence of 2000 X molar excess of insecticides
or 1000 X molar excess of TCDF. As shown in Fig. 4 and surprisingly, under these experimental conditions, none of these compounds was able to completely displace [3H]TCDD from its binding sites, suggesting that they would not be a ligand of the AhR. These results do not exclude an interference with the AhR, but suggest that direct interaction does not occur between unchanged insecticides and AhR.
4. Conclusions Several conclusions could be drawn from these studies: (1) the induction of cytochromes P4501A1 appeared to be much more sensitive than cytotoxicity test for determining the biological effects of pesticides; (2) there are significant differences between the cell types and the derivative. Indeed, carbaryl was shown to be an inducer in all cell types, whereas cypermethrin and tetrachlorvinphos are inducers only in hepatocytes. DFU and TCV are inducers in hepatocytes, but inhibitors in HepG2 and HaCaT cells; (3) significant interspecies variability exists concerning induction which seems more important in rat than in human cells; (4) finally, there is no direct interaction of the tested insecticides with the Ah receptor, which would imply, either a AhR-independent activation of CYP1A1 by these or metabolites formation which in turns follows the classical AhR transduction pathway. However, a number of questions remain yet unanswered:—what is the origin of the inter-tissue or inter-species differences?—is there a relationships between enzyme induction, and toxicity?—what are specific enzymes involved in pesticides metabolism in mammals?—are there chemical interactions between pesticides and other environmental pollutants?… On the whole, although insecticides concentrations in the human diet are very low, the results suggest that care should be taken because of possible risk stemming from the exposure to insecticides, given the well established pharmaco-toxicological importance of CYPs induction.
C. Delescluse et al. / Toxicology Letters 96,97 (1998) 33–39
References Ahlborg, U.G., Brouwer, A., Fingerhut, M.A., Jakobson, J.L., Jakobson, S.W., Kennedy, S.W., Kettrup, A.A., Koeman, J.H., Poiger, H., Rappe, C., Safe, S.H., Seegal, R.F., Tuomisto, J., Van den Berg, M., 1992. Impact of polychlorinated dibenzo-p-dioxins, dibenzofurans, and biphenyls on human and environmental health, with special emphasis on application of the toxic equivalency factor concept. Eur. J. Pharmacol. 228, 179–199. Berry, M.N., Friend, D.S., 1969. High-yield preparation of isolated rat liver parenchymal cells. J. Cell. Biol. 43, 506– 520. Boukamp, P., Petrussevska, R.T., Breitkreutz, D., Hornung, J., Markham, A., Fusenig, N.E., 1988. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J. Cell Biol. 106, 761–771. Chambers, J.E., Carr, R.L., 1995. Biochemical mechanisms contributing to species differences in insecticidal toxicity. Toxicology 105, 291 –304. Chomczynski, P., Shacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal. Biochem. 162, 156–159. de Sousa, G., Fontaine, F., Pralavorio, M., Botta-Fridlund, D., Letreut, Y., Rahmani, R., 1997. Insecticides cytotoxicity and CYP1A1/2 induction in primary human and rat hepatocytes cultures. Toxicol. in vitro 11, 451–457. Delescluse, C., Ledirac, N., de Sousa, G., Pralavorio, M., Botta-Fridlund, D., Letreut, Y., Rahmani, R., 1997. Comparative study of CYP1A1 induction by 3-methylcholanthrene in various human hepatic and epidermal cell types. Toxicol. in vitro 11, 443–450. Donato, M.T., Gomez-Lechon, M.J., Castell, J.V., 1993. A microassay for measuring cytochrome P4501A1 and P450IIB1 activities in intact human and rat hepatocytes cultured on 96-well plates. Anal. Biochem. 213, 29–33.
.
39
Dubois, M., Plaisance, H., Thome, J.P., Kremers, P., 1996. Hierarchical cluster analysis of environmental pollutants through P450 induction in cultured hepatic cells: indications for a toxicity screening test. Ecotox. Environ. Safety 34, 205 – 215. Fabre, G., Rahmani, R., Placidi, M., Combalbert, J., Covo, J., Cano, J.P., Coulange, C., Ducros, M., Rampal, M., 1988. Characterisation of midazolam metabolism using human hepatic microsomal fractions and hepatocytes in suspension obtained by perfusing whole human liver. Biochem. Pharmacol. 37, 4389 – 4397. Fautrel, A., Chesne, C., Guillouzo, A., de Sousa, G., Placidi, M., Rahmani, R., Braut, F., Pichon, J., Hoellinger, H., Vintezou, P., Diarte, I., Melcion, C., Cordier, A., Lorenzon, G., Benicourt, M., Vannier, B., Fournex, R., Peloux, A.F., Bichet, N., Gouy, D., Cano, J.P., 1991. A multicentre study of acute in vitro cytotoxicity in rat liver cells. Toxicol. in vitro 5, 543 – 547. Kurtz, P.J., Deskin, R., Harrington, R.M., 1989. Pesticides. In: Hayes, A.W. (Ed.), Principles and Methods of Toxicology, 2nd edn. Raven Press, New York, pp. 137 – 167. Ledirac, N., Delescluse, C., de Sousa, G., Pralavorio, M., Lesca, P., Amichot, M., Berge, J.B., Rahmani, R., 1997. Carbaryl induces CYP1A1 gene expression in HepG2 and HaCaT cells but is not a ligand of the human hepatic Ah receptor. Toxicol. Appl. Pharmacol. 144, 177 – 182. Lesca, P., Fernandez, N., Roy, M., 1987. The binding components for 2,3,7,8-tetrachlorodibenzo-p-dioxin and polycyclic aromatic hydrocarbons. J. Biol. Chem. 262, 4827 – 4835. Reiners, J.J., Cantu, A.R., Pavone, A., Smith, S.C., Gardner, C.R., Laskin, D.L., 1990. Fluorescence assay for per-cell estimation of cytochrome P-450-dependent monooxygenase activities in keratinocytes suspensions and cultures. Anal. Biochem. 188, 317 – 324. Safe, S.H., 1986. Comparative toxicology and mechanism of action of polychlorinated dibenzo-p-dioxins and dibenzofurans. Annu. Rev. Pharmacol. Toxicol. 26, 371 – 399.
Cytotoxic effects and induction of cytochromes P450 1A1/2 by insecticides, in hepatic or epidermal cells: binding capability to the Ah receptor C. Delescluse a,*, N. Ledirac a, G. de Sousa a, M. Pralavorio a, P. Lesca b, R. Rahmani a a
Laboratoire de Pharmaco-toxicologie Cellulaire et Mole´culaire, Centre de Recherches INRA, 41 Bd du Cap, 06606 Antibes, France b Laboratoire de Pharmaco-toxicologie, Centre INRA, B.P. 3, 31931 Toulouse, France
Abstract Insecticides deserve particular attention since the general population is potentially exposed to such chemicals through many routes. We therefore tested the comparative acute and chronic toxicity of chemicals belonging to the major insecticides families (DDT, malathion and tetrachlorvinphos, carbaryl, cypermethrin, diflubenzuron), in hepatocytes, HepG2 and HaCaT cell lines. Two kinds of end-points were used: cytotoxicity parameters and CYP1A1 induction. Except for cypermethrin and diflubenzuron, all these chemicals exerted a cytotoxic effect in hepatocytes and HaCaT, but not in HepG2 cells. However, the induction of the EROD activity appeared more sensitive since a response was detected at lower concentrations. Significant differences were observed between the cell types and the insecticides. Furthermore, these chemicals were unable to displace [3H]TCDD from its binding sites, suggesting that they would not be a ligand of the Ah receptor. The experimental approach used herein may be a good means for predicting the acute and chronic toxicity of pesticides. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Insecticides; CYP1A1 induction; Cytotoxicity; Ah receptor
Abbre6iations: BSA, bovine serum albumin; DMEM, Dulbecco modified Eagle’s Medium; CBR, carbaryl; CPR, cypermethrin; CYP1A1, cytochrome P4501A1; DFU, diflubenzuron; EROD, ethoxyresorufin-O-deethylase; FCS, fetal calf serum; MAL, malathion; 3-MC, 3-methylcholanthrene; NADP, nicotinamide adenine dinucleotide phosphate; PBS, phosphate buffer solution; TCDD, 2,3,7,8-tetrachlorodibenzop-dioxin; TCDF, tetrachlorodibenzofuran; TCV, tetrachlorvinphos. * Corresponding author.
1. Introduction Pesticides represent high volume and widely used environment chemicals. Annual commercial production averages 5×108 kg in the USA, with 1800 chemicals registered so far into 22000 different formulations and a retail sales value of several billions dollars. They contrast with other chemical classes such as occupational hazards or
0378-4274/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0378-4274(98)00047-2
34
C. Delescluse et al. / Toxicology Letters 96,97 (1998) 33–39
therapeutic drugs, in that they necessarily are toxic to selected biological systems and are deliberately deposited into the environment. Moreover, due to their lipophilic nature and slow chemical and biological degradation, these molecules tend to be taken up by biological membranes and tissues, concentrated in organisms, and progress up the food chain (Safe, 1986; Ahlborg et al., 1992; Dubois et al., 1996). This bioaccumulation and the resultant constant exposure either via direct exposure or food consumption and drinking water may cause a gradual deterioration of health. There is therefore a continuous debate concerning their possible role in various chronic effects in human, including neurotoxicity, development and reproductive effects, cyto- and genotoxicity, enzyme induction… (Kurtz et al., 1989; Chambers and Carr, 1995). In that context, we aimed at assessing the acute and chronic toxicity of various compounds belonging to the major insecticides families: DDT for chlorinated hydrocarbons, malathion (MAL) and tetrachlorvinphos (TCV) for organophosphorus, carbaryl (CBR) for carbamates, cypermethrin (CPR) for pyrethroids and diflubenzuron (DFU) for benzoylurea. As the main potential portals of entry of insecticides are the skin (in case of dermal contact) and the liver (in case of ingestion), two kinds of cellular models were used: (i) epidermal cells such as spontaneously immortalised keratinocytes HaCat cell line (Boukamp et al., 1988); (ii) hepatic cells such as human and rat hepatocytes, as well as the HepG2 human hepatoma cell line. These models were also selected because they possess the enzyme equipment to bioactivate or detoxify xenobiotics (Delescluse et al., 1997). As concerns toxicological end-points, different kinds of parameters were used: (i) cytotoxicity parameters, namely MTT and neutral red tests for mitochondrial and lysosomal functions respectively; (ii) parameters measuring the chronic effects of xenobiotics, such as the induction of cytochrome P450 1A1/2 expression which is considered to be one of the most sensitive and specific cellular response to harmful xenobiotics. The current study highlight the interest of using various cell types and end-points for evaluating or predicting the potential acute and chronic toxicity of environmental pollutants.
Table 1 Comparative in vitro cytotoxicity of insecticides determined by the MTT test, after 48 h exposure I.C.50 (mM)
DDT MAL TCV CBR CPR DFU
Hepatocytes
HepG2
HaCaT
250 253 40 264 \1000 \1000
\100 \100 \100 \100 \100 \100
70 20 77 \100 \100 \100
Results are representative of experiments using HaCaT cells and human hepatocytes (mean of five replicate). Cells were treated for 48 h with various concentrations of pesticides and the MTT test was performed as described in Section 2. Pesticide solutions were prepared in DMSO, and the final concentration of DMSO never exceeded 0.5% (v/v).
2. Materials and methods
2.1. Chemicals Dulbecco modified Eagle’s medium (DMEM), penicillin–streptomycin, L-glutamine, sodium pyruvate, Eagle’s non-essential amino-acids, fetal calf serum (FCS), were from Bioproducts (Germany). William’s medium, DMSO, glucose-6-
Fig. 1. In vitro cytotoxicity of insecticides as determined by the MTT test, after 48 h of exposure. Results are representative of experiments using human hepatocytes ( — ) and spontaneously immortalized keratinocytes, HaCaT cells ( – – – ). Each point is the mean of at least five replicates.
C. Delescluse et al. / Toxicology Letters 96,97 (1998) 33–39
35
Fig. 2. Maximal inducing effect of the different insecticides on rat hepatocytes (HEP), HepG2 and HaCaT cells, by reference to 3-MC. EROD activity was determined as described in Section 2. Results are expressed as the means 9S.D. for at least two independent experiments in triplicate. Chemical concentrations: 3-MC (0.5 mM), MAL (50 mM), DDT (10 mM), CBR (100 mM), CPR (100 mM), DFU (100 mM), TCV (75 mM). Asterisks indicate significant differences from control (*, P B0.05; **, P B0.001; one-way ANOVA and Newman–Keul’s post hoc test).
phosphate, glucose-6-phosphate dehydrogenase, b-NADP, 3-methycholanthrene (3-MC) were from Sigma (France). 7-ethoxyresorufin and resorufin were from Boehringer (Germany). Insecticides were from Cluzeau Info Labo (France). 2,3,7,8-tetrachlorodibenzofuran (TCDF) was from Chemsyn Sciences Lab (USA). Hydrocortisone hemisuccinate was from Roussel-Uclaf.
2.2. Cell culture and treatment Human hepatocytes were obtained from liver biopsies resected from secondary tumors, by the classical two-step collagenase perfusion technique (Fabre et al., 1988). Rat hepatocytes were isolated, as previously described (Berry and Friend, 1969), from male Sprague – Dawley weighing 220 – 240 g. Freshly isolated cells were resuspended in William’s medium containing 10% FCS and supplemented with penicillin (50 U/ml), streptomycin (50 mg/ml), and insulin (0.1 U/ml). Hepatocytes were seeded in collagen type I-coated dishes. Plates containing hepatocytes were incubated 4 h at 37°C under a humidified 5% CO2 atmosphere. The medium was then renewed with the same
initial medium without FCS but supplemented with hydrocortisone hemisuccinate (1mM), and containing increasing concentrations of the different pesticides. HepG2 and HaCaT were cultured in DMEM supplemented with 10% FCS, penicillin (100 U/ ml), streptomycin (100 mg/ml), sodium pyruvate (1 mM), non-essential amino acids (0.1 mM) and L-glutamine (2 mM). Cell cultures were subcultured every 4 days for HaCaT and 6 days for HepG2 at 1:10 and 1:2 ratios, respectively. Cells were seeded on 100 mm diameter plates for Northern blot analysis, or 96-well microtiter plates for EROD. Then, insecticides or 3-MC, dissolved in DMSO (final concentration 0.5%) were added to the cultures for 72 h, with a change of medium every 24 h.
2.3. Cytotoxicity test The cytotoxic effects of the insecticides were assessed after 48 or 72 hr of exposure, by using the MTT test for hepatocytes and cell lines, respectively. The assays were carried out as previously described (Fautrel et al., 1991).
36
C. Delescluse et al. / Toxicology Letters 96,97 (1998) 33–39
2.4. EROD (7 -ethoxyresorufin-O-deethylase) acti6ity assay The determination of the EROD enzyme activities in cells cultured for 3 days with the test compounds, was performed according to Reiners et al. (1990), Donato et al. (1993), with slight modifications. Briefly, after treatment, the medium was discarded and 25 ml of TMB buffer (Tris –HCl 100 mM pH 7.8, MgCl2 2.5 mM, BSA 0.06%) containing 1% glycerol was added per well and the cultures were frozen at −80°C. After thawing, 200 ml/well of TMB buffer containing glucose-6-phosphate (3 mM), NADP (0.5 mM), dicoumarol (10 mM), ethoxyresorufin (2 mM) and glucose-6-phosphate dehydrogenase (0.1 U/ml) were added. The EROD activity was measured at 37°C by spectrofluorimetry (lex =600 nm, lem = 535 nm) by following the kinetics of appearance of resorufin from ethoxyresorufin.
2.5. mRNA analysis Three days after treament, total RNA was isolated from the cell culture by the acidic phenol extraction procedure (Chomczynski and Shacchi, 1987). Twenty micrograms of RNA were sizefractionated on a 0.9% agarose gel containing 10% formaldehyde and transferred to a nitro-cellulose membrane. Hybridization was performed with 476 pb cDNA insert of human CYP1A1 mRNA corresponding to nucleotides +311 to +787 and labelled using 32P.
2.6. Ligand binding Three hundred microliters of enriched 9S fraction containing human Ah receptor were incubated with 40 nM of [3H]TCDD (2,3,7,8tetrachlorodibenzo-p-dioxin) in absence or presence of either 40 mM TCDF or 80 mM insecticides. [3H]TCDD, TCDF, (tetrachlorodibenzofuranin) and insecticides were dissolved in DMSO. Then, cytosol samples were analysed by velocity sedimentation in sucrose gradient (10– 30%). Gradients were centrifuged at 4°C for 2 h (372000 g), and 22 fractions (282 ml; eight drops per fraction) were collected. The radioactivity of each fraction was determined by liquid scintillation counting (Lesca et al., 1987).
2.7. Statistical analysis
Fig. 3. Induction of CYP1A1/2 mRNA in cultures of human hepatocytes, HepG2 cells and HaCaT cells. Cells were treated either with DMSO or various concentrations of chemicals for 72 h, with a change of medium every day. 10 mg (hepatocytes) or 20– 30 mg (HepG2 and HaCaT) of total RNA was analysed and CYP1A1/2 mRNAs were revealed with a radiolabeled CYP1A1/2 probe as described in Section 2. Chemical concentrations: (lane 1) DMSO 0.5%; (lane 2) 3-MC 2 mM (hepatocytes) and 1 mM (HepG2 and HaCaT cells); (lane 3) DFU 50 mM; (lane 4) TCV 25 mM (hepatocytes) and 50 mM (HepG2 and HaCaT cells); (lanes 5) DDT 10 mM (hepatocytes) and 50 mM (HepG2 and HaCaT cells); (lane 6) CBR 75 mM.
All enzymatic results were evaluated using a one-way analysis of variance for control and insecticide-treated cells. The statistical significance was calculated using Newman–Keuls post hoc test for multiple mean comparisons against the control set. Cytoxicity data were standardized as the percentage of control cell viability.
3. Results and Discussion
3.1. Cytotoxicity Cell viability was estimated by the MTT test. As shown in Table 1 which reports the comparative cytotoxicity (IC50 values) for the various in-
C. Delescluse et al. / Toxicology Letters 96,97 (1998) 33–39
37
Fig. 4. Absence of competitive binding of various insecticides on the Ah receptor. The enriched fraction from human hepatocyte cytosols were incubated with 40 nM [3H]TCDD, in absence or presence of 80 mM insecticides or 40 mM TCDF, for 2 h at 4°C. Then samples were analysed by velocity sedimentation on 10–30% sucrose gradients as described in Section 2.
secticides on different cell types, significant differences were observed between the tested compounds. Except cypermethrin and diflubenzuron, all these chemicals exerted a cytotoxic effect with a IC50 ranging from 20 to 264 mM in hepatocytes and HaCaT, but not in HepG2 cells. Tetrachlorvinphos was the most toxic derivative for human hepatocytes whereas it was malathion in
HaCaT cell line. However, it should be pointed out that due to the poor solubility of some of the insecticides in culture medium, IC50s may be under-estimated. Fig. 1 illustrates the dose-dependent toxic effect of the three most toxic insecticides and confirms that hepatocytes and HaCaT have different sensitivity toward these compounds.
38
C. Delescluse et al. / Toxicology Letters 96,97 (1998) 33–39
3.2. CYP1A1 induction The ability of the six insecticides in inducing EROD activity was tested in human and rat hepatocytes, HepG2 and HaCaT cell lines, at various non cytotoxic concentrations. Fig. 2 shows the maximal inducing effect of the insecticides in rat cells, by reference to the positive control 3-MC. This test appeared much more sensitive than cytotoxic tests, since a response to insecticides was detected at concentrations as low as 10 mM. Moreover, significant differences were observed between the cell types. Four compounds, namely carbaryl, cypermethrin, diflubenzuron and tetrachlorvinphos (Fig. 2) were shown to significantly activate the EROD activity: from 6 to 9-fold-over control DMSO treated cells in hepatocytes. The effect of the strongest inducers: diflubenzuron and tetrachlorvinphos was dose-dependent (de Sousa et al., 1997). These results were confirmed by northern blot analysis for diflubenzuron, but surprisingly not for tetrachlorvinphos (Fig. 3). This observation could be explained by a more rapid degradation of mRNAs following tetrachlorvinphos treatment, or by different regulation mechanisms for CYP1A1/2 gene expression by the two compounds. Different observations were done in HaCaT and HepG2 cells, where only carbaryl was able to significantly induce the EROD activity (Fig. 2). This effect is dosedependent in both cell types (Ledirac et al., 1997) and is confirmed by northern blot analysis of CYP1A1 mRNAs (Fig. 3). On the other hand, diflubenzuron and tetrachlorvinphos inhibit basal EROD activity (Figs. 2 and 3) in these cell lines whereas they were inducers in hepatocytes.
3.3. Binding capability to the Ah receptor To further confirm these results, we examined the capability of these chemicals to bind the human hepatic Ah receptor (AhR). Competitive binding assays were performed by incubating the 9S enriched fraction of human liver cytosol with [3H]TCDD at 40 nM, in absence or presence of 2000 X molar excess of insecticides
or 1000 X molar excess of TCDF. As shown in Fig. 4 and surprisingly, under these experimental conditions, none of these compounds was able to completely displace [3H]TCDD from its binding sites, suggesting that they would not be a ligand of the AhR. These results do not exclude an interference with the AhR, but suggest that direct interaction does not occur between unchanged insecticides and AhR.
4. Conclusions Several conclusions could be drawn from these studies: (1) the induction of cytochromes P4501A1 appeared to be much more sensitive than cytotoxicity test for determining the biological effects of pesticides; (2) there are significant differences between the cell types and the derivative. Indeed, carbaryl was shown to be an inducer in all cell types, whereas cypermethrin and tetrachlorvinphos are inducers only in hepatocytes. DFU and TCV are inducers in hepatocytes, but inhibitors in HepG2 and HaCaT cells; (3) significant interspecies variability exists concerning induction which seems more important in rat than in human cells; (4) finally, there is no direct interaction of the tested insecticides with the Ah receptor, which would imply, either a AhR-independent activation of CYP1A1 by these or metabolites formation which in turns follows the classical AhR transduction pathway. However, a number of questions remain yet unanswered:—what is the origin of the inter-tissue or inter-species differences?—is there a relationships between enzyme induction, and toxicity?—what are specific enzymes involved in pesticides metabolism in mammals?—are there chemical interactions between pesticides and other environmental pollutants?… On the whole, although insecticides concentrations in the human diet are very low, the results suggest that care should be taken because of possible risk stemming from the exposure to insecticides, given the well established pharmaco-toxicological importance of CYPs induction.
C. Delescluse et al. / Toxicology Letters 96,97 (1998) 33–39
References Ahlborg, U.G., Brouwer, A., Fingerhut, M.A., Jakobson, J.L., Jakobson, S.W., Kennedy, S.W., Kettrup, A.A., Koeman, J.H., Poiger, H., Rappe, C., Safe, S.H., Seegal, R.F., Tuomisto, J., Van den Berg, M., 1992. Impact of polychlorinated dibenzo-p-dioxins, dibenzofurans, and biphenyls on human and environmental health, with special emphasis on application of the toxic equivalency factor concept. Eur. J. Pharmacol. 228, 179–199. Berry, M.N., Friend, D.S., 1969. High-yield preparation of isolated rat liver parenchymal cells. J. Cell. Biol. 43, 506– 520. Boukamp, P., Petrussevska, R.T., Breitkreutz, D., Hornung, J., Markham, A., Fusenig, N.E., 1988. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J. Cell Biol. 106, 761–771. Chambers, J.E., Carr, R.L., 1995. Biochemical mechanisms contributing to species differences in insecticidal toxicity. Toxicology 105, 291 –304. Chomczynski, P., Shacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal. Biochem. 162, 156–159. de Sousa, G., Fontaine, F., Pralavorio, M., Botta-Fridlund, D., Letreut, Y., Rahmani, R., 1997. Insecticides cytotoxicity and CYP1A1/2 induction in primary human and rat hepatocytes cultures. Toxicol. in vitro 11, 451–457. Delescluse, C., Ledirac, N., de Sousa, G., Pralavorio, M., Botta-Fridlund, D., Letreut, Y., Rahmani, R., 1997. Comparative study of CYP1A1 induction by 3-methylcholanthrene in various human hepatic and epidermal cell types. Toxicol. in vitro 11, 443–450. Donato, M.T., Gomez-Lechon, M.J., Castell, J.V., 1993. A microassay for measuring cytochrome P4501A1 and P450IIB1 activities in intact human and rat hepatocytes cultured on 96-well plates. Anal. Biochem. 213, 29–33.
.
39
Dubois, M., Plaisance, H., Thome, J.P., Kremers, P., 1996. Hierarchical cluster analysis of environmental pollutants through P450 induction in cultured hepatic cells: indications for a toxicity screening test. Ecotox. Environ. Safety 34, 205 – 215. Fabre, G., Rahmani, R., Placidi, M., Combalbert, J., Covo, J., Cano, J.P., Coulange, C., Ducros, M., Rampal, M., 1988. Characterisation of midazolam metabolism using human hepatic microsomal fractions and hepatocytes in suspension obtained by perfusing whole human liver. Biochem. Pharmacol. 37, 4389 – 4397. Fautrel, A., Chesne, C., Guillouzo, A., de Sousa, G., Placidi, M., Rahmani, R., Braut, F., Pichon, J., Hoellinger, H., Vintezou, P., Diarte, I., Melcion, C., Cordier, A., Lorenzon, G., Benicourt, M., Vannier, B., Fournex, R., Peloux, A.F., Bichet, N., Gouy, D., Cano, J.P., 1991. A multicentre study of acute in vitro cytotoxicity in rat liver cells. Toxicol. in vitro 5, 543 – 547. Kurtz, P.J., Deskin, R., Harrington, R.M., 1989. Pesticides. In: Hayes, A.W. (Ed.), Principles and Methods of Toxicology, 2nd edn. Raven Press, New York, pp. 137 – 167. Ledirac, N., Delescluse, C., de Sousa, G., Pralavorio, M., Lesca, P., Amichot, M., Berge, J.B., Rahmani, R., 1997. Carbaryl induces CYP1A1 gene expression in HepG2 and HaCaT cells but is not a ligand of the human hepatic Ah receptor. Toxicol. Appl. Pharmacol. 144, 177 – 182. Lesca, P., Fernandez, N., Roy, M., 1987. The binding components for 2,3,7,8-tetrachlorodibenzo-p-dioxin and polycyclic aromatic hydrocarbons. J. Biol. Chem. 262, 4827 – 4835. Reiners, J.J., Cantu, A.R., Pavone, A., Smith, S.C., Gardner, C.R., Laskin, D.L., 1990. Fluorescence assay for per-cell estimation of cytochrome P-450-dependent monooxygenase activities in keratinocytes suspensions and cultures. Anal. Biochem. 188, 317 – 324. Safe, S.H., 1986. Comparative toxicology and mechanism of action of polychlorinated dibenzo-p-dioxins and dibenzofurans. Annu. Rev. Pharmacol. Toxicol. 26, 371 – 399.