Arch Toxicol (2000) 73: 588±593

Ó Springer-Verlag 2000

O R G A N T O X IC IT Y A N D M E C H A N I S M S

é. A. Voie á F. Fonnum

Effect of polychlorinated biphenyls on production of reactive oxygen species (ROS) in rat synaptosomes

Received: 21 June 1999 / Accepted: 14 September 1999

Abstract In this paper the e€ect of polychlorinated biphenyls (PCBs) on the production of reactive oxygen species (ROS) in rat synaptosomes is elucidated. The e€ect of methylmercury (MeHg) on rat synaptosomes was included as a positive control since several studies have investigated the ability of this substance to produce ROS. The exposure of the synaptosomes to the congener 2,2-dichlorobiphenyl (2,2¢-DCB; 12.5 lM) produced a linear increase in the formation of 2¢,7¢-dichloro¯uorescein (DCF) as a measure for the production of ROS. The congeners 2,2¢-DCB (12.5 lM) and 3,3¢-DCB (12.5 lM) stimulated, as expression of ROS production, a signi®cant increase in DCF formation formation compared to the control. The congeners 2-chlorobiphenyl (2-CB) and 2,2¢,6-trichlorobiphenyl (2,2,6¢-TCB) were active at 50 lM, whereas 2,2¢,4,4¢,5,5¢-hexachlorobiphenyl (2,2¢,4,4¢,5,5¢-HCB), 4,4¢-DCB and 2,2¢,6,6¢tetrachlorobiphenyl (2,2¢,6,6¢-TeCB) were not active at this concentration. The increased formation of ROS in response to 2,2¢-DCB and MeHg in the synaptosomes was dependent on extracellular Ca2+. A phospholipase C inhibitor, U73122, was shown to signi®cantly decrease the ROS formation induced by 2,2¢-DCB, but did not reduce the ROS formation induced by MeHg. Ethanol (1%), a phospholipase D modulator, reduced the ROS formation induced by MeHg and by 2,2¢-DCB by 33 and 52%, respectively. Wortmannin (25 nM), an inhibitor of phosphatidylinositol 3-kinase, completely inhibited the ROS formation induced by MeHg and 2,2¢-DCB. It appears that the ROS-stimulating PCBs are the same congeners found to be neuroactive in other types of study. Phospholipase C and D and phosphatidylinositol 3-kinase seem to be involved in the intracellular signalling system that leads to ROS formation during PCB exposure. é.A. Voie (&) á F. Fonnum Norwegian Defence Research Establishment, Division for Environmental Toxicology, P.O. Box 25, 2027 Kjeller, Norway Tel: +47-63807824; Fax: +47-63807811

Key words Polychlorinated biphenyls (PCB) á Synaptosomes á Reactive oxygen species (ROS) á Free radicals á Mechanism

Introduction Polychlorinated biphenyls (PCBs) have been used as isolating and cooling liquids in electrical components. These compounds show a complex spectrum of biological and toxicological properties. On account of being lipophilic and widely distributed, PCBs accumulate in organisms and are bioconcentrated in the food chain (Van den Berg et al. 1993). Several seabirds and marine mammals are heavily contaminated with PCBs and are believed to su€er from the toxic e€ects of these compounds (Norstrom and Muir 1994). Much of the research on PCBs and their toxicity has focused on e€ects such as cytochrome P450 1A1 induction, hepatomegaly, thymic atrophy and reduced body weight gain, all of which are mediated by coplanar PCB congeners through a common receptor, the aryl hydrogen (Ah)-receptor (Safe 1990; Nebert 1989). Less attention has been paid to ortho-substituted nonplanar PCB congeners. Recently it was shown that these congeners, which are abundantly present in the environment, are neurotoxicants (Kodavanti et al. 1993, 1994, 1995; Seegal et al. 1990). Epidemiological data show that polychlorinated biphenyls have neurotoxicological e€ects on humans (Rogan and Gladen 1992; Tilson et al. 1990; Jacobson et al. 1990). However, there is no correlation between the coplanar, Ah-mediated physical signs of exposure (for example, chloracne and hyperpigmentation), and the observed neurotoxicologic e€ects. This suggests that these e€ects may be caused by exposure to the noncoplanar, ortho-substituted PCB congeners present in many commercial mixtures of PCBs, contaminated food products and breast milk (Seegal 1996). Seegal et al. (1991), found that ortho-substituted PCB congeners were responsible for reducing brain dopamine levels. ortho-Substituted PCB congeners also increase intracel-

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lular calcium concentration and interfere with intracellular signalling systems in cerebellar granule cells and granulocytes (Kodavanti et al. 1993, 1994; Voie and Fonnum 1998). The activation of the intracellular signalling system in human granulocytes leads to stimulation of the respiratory burst and an increased production of reactive oxygen species (ROS; Voie et al. 1998). ROS are identi®ed as key participants in brain damage resulting from N-methyl-D-aspartic acid (NMDA) receptor-mediated damage (Fonnum 1998). At low concentrations ROS may trigger intracellular signalling transduction pathways in di€erent cell types (Chen et al. 1995; Bae et al. 1997). Hydrogen peroxide also has the ability to cause marked functional changes in isolated nerve endings: it induces oxidative stress, [Na+]i and [Ca2+]i elevation, plasma membrane depolarization, enhances Ca2+ in¯ux via voltage dependent channels and decreases the intracellular ATP level (Tretter and Adam-Vizi 1996; Tretter et al. 1997). Recently the production of ROS has been detected in a variety of cells which have been stimulated by cytokines such as transforming growth factor-b1, interleukin-1, and tumour necrosis factor-a, with peptide growth factors such as platelet degranulation factor (PDGF) and NF-jB, or with agonists of receptors with seven transmembrane spans such as lysophosphatidic acid and angiotensin II (Chen et al. 1995; Bae et al. 1997). This paper aims to investigate the e€ects of PCBs on the production of ROS in rat synaptosomes. Hydrogen peroxide, as one of the reactive oxygen species may be measured with the ¯uorescent probe 2¢,7¢-dichloro¯uorescein diacetate (DCFH-DA; Oyama et al. 1994; LeBel et al. 1992).

30 min. The pellet (P2) containing synaptosomes and mitochondria was resuspended in 0.32 M sucrose and used as such. For preparation of puri®ed synaptosomes (P2B fraction), the homogenate was centrifuged at 1000 ´g for 10 min, and the supernatant was further centrifuged at 20,000 ´g for 30 min. The pellet (P2) was resuspended in 0.32 M sucrose, layered onto a discontinuous sucrose gradient consisting of 1.2 M and 0.8 M sucrose and centrifuged in a swing-out rotor at 65,000 ´g for 30 min. The synaptosomes were collected from the top of the 1.2 M sucrose layer and the mitochondria in the pellet beneath the 1.2 M sucrose layer. Exposure to PCBs Stock solutions were prepared by dissolving the PCB congeners in methanol. The ®nal concentration of methanol was 1%. This concentration of methanol did not in¯uence signi®cantly the normal ROS production. The synaptosomes were diluted to 0.43 ‹ 0.078 (n ˆ 3) mg protein/ml. The protein concentration was determined using the method described by Lowry et al. (1951). Assay of the formation of hydrogen peroxide The formation of hydrogen peroxide was determined by the procedure described by LeBel and Bondy (1990). The chemical compound DCFH-DA is freely permeable across cell membranes and is incorporated into hydrophobic lipid regions of the cell. The acetate moiety is cleaved o€ by cellular esterases leaving the non¯uorescent DCFH. Hydrogen peroxide and peroxidases oxidize the DCFH to DCF, which is ¯uorescent (530 nm; LeBel et al. 1992). Synaptosomes were diluted in 5 ml HBSS containing 20 mM HEPES and 5 mM glucose and incubated for 15 min at 34 °C together with 10 lM DCFH-DA in methanol. Dye-loaded samples were centrifuged at 9000 ´ g for 8 min at 4 °C and the pellets were resuspended in 5 ml of HBSS bu€er. The synaptosomes (100 ll of 1 mg/ ml suspension) were added to a microplate and the measurement of DCF-mediated ¯uorescence was performed on a computerized Perkin-Elmer LS50 luminescence spectrometer, using excitation wavelength 488 nm and emission wavelength 525 nm. The incubation temperature was 37 °C. The ®nal volume of the reaction mixture was 250 ll.

Materials and methods

Statistical analysis

Chemicals

Data were analysed by Students t-test.

The ¯uorescent probes fura-2 and DCFH-DA were purchased from Molecular Probes. The congeners 2-chlorobiphenyl, 2,2¢-dichlorobiphenyl, 2,3¢-dichlorobiphenyl, 3,3¢- dichlorobiphenyl, 4,4¢-dichlorobiphenyl, 2,2¢,6-trichlorobiphenyl, 2,2¢,6,6¢-tetrachlorobiphenyl and 2,2¢,4,4¢5,5¢-hexachlorobiphenyl were purchased from the company Dr Ehrenstorfer (Germany). Wortmannin was purchased from Sigma Chemical Co. U-73122 and 3-[1-(3-dimethylaminopropyl)-indol-3-yl]-3-(indol-3-yl)-maleimide (bisindolylmaleimide) were purchased from Calbiochem. Hank's Balanced Salts Solution (HBSS) and HEPES bu€er were purchased from GibcoBRL. Preparation of synaptosomes Male Wistar rats of 200±250 g body wt. were purchased from Mùllegaard breeding laboratories, Denmark. The animals were decapitated and the brain was dissected out and homogenized in 0.32 M sucrose at 4 °C. Synaptosomes were prepared according to the method of Gray and Whittaker (1962). Both crude (P2 fraction) and puri®ed (P2B fraction) synaptosomes were prepared. For preparation of crude synaptosomes, the homogenate was centrifuged at 1000 ´g for 10 min, and 1.6 M sucrose was added to the supernatant until the solution reached a sucrose concentration of 0.8 M. The supernatant was further centrifuged at 20,000 ´g for

Results Rat crude synaptosomes were exposed to the PCB congener 2,2¢-DCB (12.5 lM) and the formation of ROS was measured with the ¯uorescent probe DCFH. The exposure to 2,2¢-DCB produced a linear increase in DCF formation as a measure of ROS production (Fig. 1). A small increase in the formation of ROS was observed with a concentration as low as 3 lM 2,2¢-DCB, while the formation of ROS obtained with 12.5 lM did not di€er from the formation of ROS obtained with 50 lM 2,2¢-DCB. Not all PCB congeners were active. There was a signi®cant di€erence between the control and the congeners 2,2¢-DCB (12.5 lM, P ˆ 0.006) and 3,3¢-DCB (12.5 lM, P < 0.03; Fig. 2). The congeners 2-CB and 2,2¢,6-TCB (50 lM) increased ROS production by 193 and 195% respectively, whereas 2,2¢,4,4¢,5,5¢-HCB, 4,4¢-DCB and 2,2¢,6,6¢-TeCB were not active at 50 lM (data not shown).

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Fig. 1 2¢,7¢-Dichloro¯uorescein (DCF) formation as an expression for the formation of reactive oxygen species (ROS) in a crude rat synaptosome fraction (P2) during exposure to MeHg (50 lM) and 2,2¢-dichlorobiphenyl (2,2¢-DCB; 12.5 lM) Data are mean ‹ SD, nˆ3

Fig. 2 Relative rates of DCF formed as a measure of the formation of ROS in a crude rat synaptosome fraction (P2) during exposure to di€erent polychlorinated biphenyl (PCB) congeners (12.5 lM). All values are relative to control (100%). Data are mean ‹ SD (bars), values from three di€erent synaptosomal preparations. See the Materials and methods for details of the preparation of the synaptosome fractions

The crude synaptosome fraction was separated on a density gradient into myelin, synaptosomes and mitochondria. The purity of the fraction was indicated by the synaptosome marker choline acetyltransferase (ChaT), the `free' mitochondrial marker c-aminobutyric acidtransaminase (GABA-T) and the general mitochondrial

marker fumarase (Fonnum 1968). The term `free' mitochondria relates to mitochondria in the fraction which is not included inside any cell/synaptosome. Fumarase, localised to both synaptosomal mitochondria and free mitochondria (Fonnum 1968), was found in equal concentrations in the synaptosomal and the mitochondrial fractions (Table 1). All three fractions were incubated with DCF, stimulated with MeHg (50 lM) and 2,2¢DCB (50 lM) and the production of ROS was measured. The synaptosomal fraction produced ROS upon stimulation with MeHg and 2,2¢-DCB (P < 0.01; Fig. 3). The mitochondrial fraction responded signi®cantly upon stimulation by MeHg (P < 0.01) and 2,2¢DCB (P < 0.05; Fig. 3). To elucidate whether the elevated production of ROS is associated with synaptosomes or free mitochondria in the fraction, an experiment with mitochondria from rat liver was performed. The experiment showed that contrary to MeHg (P < 0.0001) 2,2¢-DCB is a poor inducer of ROS in rat liver mitochondria (Fig. 4). Of the congeners studied, it was found that 2,2¢-DCB was the most e€ective, and also has been well studied in other experiments (Kodavanti et al. 1993; Voie and Fonnum, 1998; Voie et al. 1998). It was therefore selected for the studies of the mechanisms involved in ROS production of the synaptosomes. The increase in formation of ROS in response to 2,2¢-DCB and MeHg in the synaptosomes was found to be dependent on extracellular Ca2+ (Fig. 5). In the absence of extracellular Ca2+ and in the presence of 5 mM ethylene glycol-bis(baminoethyl ether)-tetraacetic acid (EGTA), the formation of ROS induced by 2,2¢-DCB and MeHg was reduced signi®cantly (P < 0.0005 and P < 0.0001, respectively; Fig. 5). Treatment of the synaptosomes with the phospholipase C inhibitor, U73122 (Naccache et al. 1993), was shown to signi®cantly decrease the formation of ROS induced by 2,2¢-DCB, (P < 0.05; Fig. 6); however U73122 did not signi®cantly reduce the formation of ROS induced by MeHg. It was found that ethanol (1%), which is a phospholipase D modulator (Bonser et al. 1989), slightly reduced the formation of ROS induced by both MeHg and 2,2¢-DCB (by 19%, P < 0.005 and 23%, P < 0.005, respectively; Fig. 6). Ethanol did not interfere with the background of ROS formation in the synaptosomes. Wortmannin, which is an inhibitor of phosphatidyl inositol 3-kinase (Naccache et al. 1993, Arcaro and Wymann 1993), signi®cantly reduced the formation of induced ROS by MeHg and 2,2¢-DCB (P < 0.0005, P < 0.0005; Fig. 6). The protein kinase C inhibitor bisindolymaleimide in a concentration of 0.5 and 5 lM did not interfere with the formation of ROS induced by 2,2¢-DCB and MeHg (data not shown).

Discussion This paper demonstrates that PCBs stimulate the production of ROS in synaptosomes. The congener 2,2¢-DCB

591 Table 1 Relative speci®c activity of marker enzymes in subcellular fractions of rat brain. (ChaT Choline acetyltransferase, GABA-T c-aminobutyric acid transaminase)

Enzyme activity

Synaptosomal fraction

Mitochondrial fraction

Myelin fraction

ChaT (lmmol/mg protein per min) GABA T (lmmol/mg protein per min) Fumarase (lmmol/mg protein per min) Protein (mg/ml)

185.9 322.8 7.6 18.4

39.4 1008.2 7.2 8.3

37.1 ‹ 3.6 88.0 ‹ 18.2 0.0 16.8 ‹ 3.4

Fig. 3 Relative rates of DCF formed as a measure of the formation of ROS in rat synaptosomes during exposure to MeHg (50 lM) and 2,2¢-DCB (50 lM). Two subcellular fractions were compared containing synaptosomes (P2B) and `free' mitochondria, respectively. All values are relative to control (100%). Data are mean ‹ SD (bars) values from three di€erent preparations. For further experimental details see the legend to Fig. 2

appears to be the most active congener (Fig. 2). The ortho-substituted congeners such as 2,2¢-DCB, 2-CB and 2,2¢,6-TCB were active, unlike the congeners 4,4¢-DCB and 2,2¢,4,4¢,5,5¢-HCB. The fully ortho-substituted congener 2,2¢,6,6¢-TeCB was inactive, whereas the metasubstituted congener 3,3¢-DCB was active (Fig. 2). These results on congeners correlated with the results on protein kinase C (PKC) translocation and the elevation of [Ca2+]i in granular cells of the rat brain (Kodavanti and Tilson 1997). In this study it was found that the ortho-substituted 2,2¢-DCB, 2,2¢,6¢-TCB and the metasubstituted congener 3,3¢-DCB were active, with 2,2¢,4,4¢,5,5¢-HCB having a small e€ect, whereas the fully ortho-substituted congener 2,2¢,6,6¢-TeCB was inactive. To elucidate the mechanism behind this e€ect, the involvement of intracellular [Ca2+] and signal transduction systems in the synaptosomes were investigated. Methylmercury, used as a positive control, similarly to 2,2¢-DCB stimulates protein phosphorylation in cerebellar granule cells, increased 45Ca2+-uptake and induced the production of inositol phosphate (Sara®an 1993).

‹ ‹ ‹ ‹

36.4 86.9 1.6 3.3

‹ ‹ ‹ ‹

10.9 98.4 4.5 0.7

Fig. 4 Relative rates of DCF formed as a measure of the formation of ROS in rat liver mitochondria during exposure to MeHg (50 lM) and 2,2¢-DCB (50 lM). All values are relative to control (100%). Data are mean ‹ SD (bars) values from three di€erent mitochondrial preparations

Results from this study indicate that the increased formation of ROS in synaptosomes is Ca2+ dependent (Fig. 5). It is known that Ca2+ is involved in production of hydrogen peroxide in synaptosomes (Zoccarato et al. 1996). Earlier studies, carried out by us indicated that 2,2¢-DCB elevates intracellular [Ca2+] and induces a respiratory burst in human granulocytes. The e€ects were reduced when the granulocytes were pre-incubated with the phospholipase C inhibitor, U-73122 (Voie and Fonnum 1998; Voie et al. 1998). This same inhibitor also reduced the formation of ROS which had been induced by 2,2¢-DCB in synaptosomes, thus indicating an involvement of phospholipase C (Fig. 6). It was discovered that 25 nM of Wortamannin, an inhibitor of phosphatidyl inositol 3-kinase, completely abolished the production of ROS induced by 2,2¢-DCB and MeHg (Fig. 6). This suggests the involvement of phosphatidyl inositol phosphates as key secondary messengers in the production of ROS. Phosphatidylinositol 3-kinase phosphorylates membrane lipids at the third position of the inositol ring producing phosphoinositides which serve as substrates for the phospholipases (Rabkin et al. 1997). Furthermore, ethanol as phospholipase D mod-

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Fig. 5 Relative rates of DCF formed as a measure of the formation of ROS in rat synaptosome fraction (P2) during exposure to MeHg (50 lM) and 2,2¢-DCB (50 lM) in presence and absence of extracellular calcium. All values are relative to control (100%). Data are mean ‹ SD (bars) values from three di€erent synaptosomal preparations

ulator (Bonser et al. 1989), reduced the 2,2¢-DCB- and MeHg-induced production of ROS (Fig. 6). The activation of phospholipase D leads to the formation of phosphatidic acid, and this may be converted to diacylglycerol (DAG; Morel et al. 1991). Protein kinase C is an enzyme which is a€ected by PCB in granular cells (Kodavanti et al. 1994; Shafer et al. 1996) and could be a key factor in the production of ROS. It has previously been shown that PKC plays a role in cholinergic-induced production of ROS in human neuroblastoma cells (Naarale et al. 1997), substance P stimulation of synovial cells (Tanabe et al. 1996), and the respiratory burst in human neutrophils stimulated by 2,2¢-DCB (Voie et al. 1998). However, results from this study show that bisindolylmaleimide (a PKC inhibitor) does not reduce ROS production in synaptosomes exposed to PCB (data not shown). It appears that ROS are produced in di€erent cell types in response to stimulation by agonists of surface receptors such as transforming growth factor-b1, interleukin-1, and tumor necrosis factor-a. Peptide growth factors such as PDGF and NF-jB, or agonists of receptors with seven transmembrane spans such as lysophosphatidic acid and angiotensin II may also be signi®cant factors. The ROS also function as signalling molecules involved in cell regulation (Chen et al. 1995; Bae et al. 1997). The activation of a membrane associated receptor by PCBs in rat cerebellar granule cells has

Fig. 6 Relative rates of DCF formed as a measure of the formation of ROS in rat synaptosome fraction (P2) during exposure to MeHg (50 lM) and 2,2¢-DCB (50 lM) in presence and absence of the phosphoinositol 3 kinase inhibitor Wortmannin (25 nM), the phospholipase C inhibitor U73122 (1 mM) and the phospholipase D modulator, ethanol (1%). See the legend to Fig. 6 for further explanation

been proposed (Kodavanti et al. 1997). The mechanism by which ROS are generated during exposure to PCB has been studied in the granulocytes (Voie and Fonnum 1998; Voie et al. 1998). In these phagocytic cells ROS are generated by the NADPH oxidase system. The mechanism by which ROS is generated in nonphagocytic cells in response to growth factors and other agonists is not known. Neither the xanthine oxidase system nor the mitochondrial respiratory chain appear to mediate receptor-triggered ROS generation (Bae et al. 1997). The mitochondrion is an organelle known to be involved in the formation of ROS, e.g. in rat hepatocytes exposed to MeHg (Atchinson and Hare 1994). The results of this study indicate that while not increasing the production of ROS in rat liver mitochondrial fractions (Fig. 4), 2,2-DCB does elevate ROS production in rat brain mitochondrial fractions (Fig. 3). It is possible that the increase in the mitochondrial fraction from the rat brain is caused by the contamination of synaptosomes. In conclusion our results indicate that some PCBs stimulate ROS producing pathways in the synaptosomes. In this respect PCB is as active as MeHg. The assay of hydrogen peroxide indicates a doubling of ROS production in synaptosomes exposed to PCBs compared

593

to the control. While these levels might not be cytotoxic, the activation of the signalling events might have other undesirable e€ects.

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