Acute toxicity of hexavalent chromium in isolated teleost hepatocytes

Aquatic Toxicology 70 (2004) 159–167

Acute toxicity of hexavalent chromium in isolated teleost hepatocytes Gerhard Krumschnabel∗ , Muhammad Nawaz ¨ Institut f¨ur Zoologie und Limnologie, Abteilung f¨ur Okophysiologie, Center for Molecular Biosciences Innsbruck (CMBI), Universit¨at Innsbruck, Technikerstraße 25, A-6020 Innsbruck, Austria Received 27 May 2004; received in revised form 10 August 2004; accepted 15 September 2004

Abstract Acute toxic effects of hexavalent chromium [Cr(VI)], a widely recognised carcinogenic, mutagenic and redox active metal, were investigated in isolated hepatocytes of goldfish (Carassius auratus). Exposure to 250 ␮M Cr(VI) induced a significant decrease of cell viability from 94% in controls to 88% and 84% after 30 min and 4 h of exposure, respectively. Cr-toxicity was associated with a concentration-dependent stimulation of the formation of reactive oxygen species (ROS). As one potential source of ROS formation we identified the lysosomal Fe2+ pool, since the ferric ion chelator deferoxamin inhibited ROS formation by approximately 15%. Lysosomal membranes remained nevertheless intact during Cr-exposure, as determined from neutral red retention in this compartment. Another significant source of ROS appear to be the mitochondria, where a presumably uncoupled increase of respiration by 20–30% was triggered by the metal. Inhibition of mitochondrial respiration by cyanide caused an approximately 40% decrease of Cr-induced ROS-formation, whereas the uncoupling agent carbonyl cyanide m-chlorophenyl hydrazine was without effect. Cellular Ca2+ homeostasis was not disturbed by Cr(VI) and thus played no role in this scenario. Overall, our data show that Cr(VI) is acutely toxic to goldfish hepatocytes, and its toxicity is associated with the induction of radical stress, presumably involving lysosomes and mitochondria as important sources of ROS formation. © 2004 Elsevier B.V. All rights reserved. Keywords: Chromium (VI); Goldfish hepatocyte; Reactive oxygen species; Calcium; Oxygen consumption; Lysosomes

1. Introduction Toxic metals are widely found in our terrestrial as well as aquatic environment. Among these metals ∗ Corresponding author. Tel.: +43 512 507 6195; fax: +43 512 507 2930. E-mail address: [email protected] (G. Krumschnabel).

0166-445X/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2004.09.001

hexavalent chromium [Cr(VI)] compounds have been found to be highly toxic and carcinogenic both in vivo and in vitro (Susa et al., 1996, 1997; Bagchi et al., 2002; Pourahmad et al., 2003). In fish, most studies on Cr toxicity employed an in vivo approach and indicated adverse effects such as a loss of lipid and glycogen stores and a decrease of protein content in vital organs (Vutukuru, 2003), loss of osmoregulatory and respiratory abilities (van der Putte et al., 1982), as well as

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inhibition of ion transporting ATPases in gills, kidney and intestinal tissue (Thaker et al., 1996). Investigations at the cellular level have been mainly made with mammalian cell models. Since liver is the main organ for Cr(VI) metabolic reduction and thus an important target site for its toxicity (Stift et al., 2000), primary cultures of hepatocytes have been amply utilised to study Cr(VI) at the cell level. Along with studies at the tissue and organism level, these investigations indicated that the induction of oxidative stress is a crucial event in the toxicity of Cr(VI). In the cell, Cr(VI) is reduced to the reactive intermediates Cr(V) and (IV), and ultimately to the more stable Cr(III) by cellular reductants (Sugiyama et al., 1991; Liu and Shi, 2001). These reduced forms of Cr may then either directly induce an increased production of reactive oxygen species (ROS) by catalysing a Fenton-like redox cycling mechanism (Shi and Dalal, 1990), or they may indirectly promote radical stress by interacting with, e.g., lysosomal and mitochondrial metabolism (Pourahmad and O’Brien, 2001). In many instances, metal toxicity is also associated with an impairment of cellular Ca2+ homeostasis, leading to an uncontrolled elevation of intracellular free Ca2+ ([Ca2+ ]i ) (Nicotera et al., 1992). This, in turn, is often paralleled by a rise of Ca2+ in the mitochondrial matrix, thereby affecting cellular oxygen consumption (Rissanen et al., 2002; Manzl et al., 2003). In this regard, the effect of Cr has received only little attention, despite of the fact that, ultimately, cell killing by redox reactive compounds may be closely intertwined with altered levels of intracellular free Ca2+ (Tan et al., 1998). In the present study we aimed at elucidating the acute toxic effects of Cr(VI) on hepatocytes from a freshwater fish, the goldfish Carassius auratus. To this end, we determined the impact of Cr(VI) exposure on several physiological parameters which are typical targets of metal toxicity in many cell models. Specifically, we investigated the potential of Cr(VI) to induce oxidative stress in these cells, i.e. its potential to cause an increase in the net formation of reactive oxygen intermediates resulting from a breakdown of cellular redoxbalance. In addition we tried to get some insight into the possible sources of ROS formation stimulated by the metal. Furthermore, in the light of the above named lack of comparable data, we studied if Cr(VI) affects Ca2+ homeostasis and oxygen consumption of goldfish

hepatocytes. Finally, we also determined the effect of Cr(VI) on lysosomal membrane stability, as the lysosomal compartment may represent both an important source as well as a target of toxic effects of various ¨ metals (Ollinger and Brunk, 1995).

2. Materials and methods 2.1. Chemicals Collagenase (Type VIII), bovine serum albumin (BSA), K2 Cr2 O7 , ionomcyin, deferoxamine mesylate (DFA), dimethyl sulfoxide (DMSO), diethyldithiocarbamate (DDTC), Vitamin E, sodium cyanide (NaCN), and carbonyl cyanide m-chlorophenyl hydrazine (CCCP) were purchased from Sigma. Fura 2-AM, 2 ,7 -dichlorofluorescin diacetate (DCF-DA), neutral red, and acridine orange were from molecular probes, all other chemicals were obtained from local suppliers. Cr(VI) was applied as K2 Cr2 O7 from a concentrated stock solution prepared in distilled water. 2.2. Animals and isolation of hepatocytes Goldfish (C. auratus) were obtained commercially and were maintained in 200 L aquaria with running water at 20 ◦ C. The animals were fed daily with a mix of trout pellets and carp flakes. Hepatocytes were isolated according to Birnbaum et al. (1976) with modifications as described previously (Krumschnabel et al., 1994). After isolation the cells were incubated for 1 h in a shaking water bath thermostated to 19 ◦ C. The standard incubation medium contained 10 mM HEPES, 135.2 mM NaCl, 3.8 mM KCl, 1.2 mM MgSO4 , 1.2 mM KH2 PO4 , 10 mM NaHCO3 , and 1.3 mM CaCl2 , pH 7.6 at 20 ◦ C, including 1% BSA. For the measurement of cytosolic free calcium [Ca2+ ]i and lysosomal stability hepatocytes (2 × 106 cells ml−1 ) were cultured in sterile, modified Leibovitz L15 medium (L15 medium plus 10 mM HEPES, 5 mM NaHCO3 , 50 ␮g ml−1 gentamycin, and 100 ␮g ml−1 kanamycin, pH titrated to 7.6) on poly-l-lysine coated coverslips. Hepatocytes were left to attach to the coverslips by over night incubation in an incubator at 19 ◦ C and 0.5% CO2 and were then washed several times with fresh standard saline without BSA (19 ◦ C, pH 7.6) before each experiment.

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2.3. Assessment of cell viability Viability of hepatocytes was assessed by the Trypan blue (0.04% w/v) exclusion method. For this purpose, cell suspensions were diluted to a concentration of 5 × 106 cells ml−1 and were incubated with or without K2 Cr2 O7 for 4 h in flasks kept in a shaking water bath at 19 ◦ C. Aliquots were taken at different times during this period and the percentage of dead and viable cells was calculated from counts on a B¨urker-T¨urk hemocytometer. Cell viability and most other parameters investigated were determined in cells exposed to 250 ␮M Cr(VI), since, as will be shown below, this concentration was the lowest to induce a significant, profound toxic effect within the time frame studied. 2.4. Cytosolic free calcium [Ca2+ ]i [Ca2+ ]i was measured in cultured hepatocytes by fluorescence microscopy as described previously (Krumschnabel et al., 2001). In brief, attached cells were incubated with 3 ␮M of the Ca2+ -sensitive fluorochrome Fura 2-AM for 60 min and were subsequently mounted in a stainless steel measuring chamber fixed on the stage of an inverted Axiovert 100 epifluorescence microscope equipped with a 40× ultraviolet objective. Subsequently, cells were washed several times with standard saline so as to remove extracellular Fura 2-AM. Cells were then covered with 1 ml of standard incubation medium and fluorescence images were taken at 1 min intervals with excitation set to 340 and 380 nm and emission detected above 510 nm. For each experiment, basal [Ca2+ ]i levels were determined by incubation of cells in standard saline for about10 min. Subsequently, for the assessment of the effect of Cr(VI) on [Ca2+ ]i , 500 ␮l of standard medium were exchanged for 500 ␮l of saline containing K2 Cr2 O7 (final concentration 250 ␮M) and the measurement continued for another 60 min. Each experiment was followed by a calibration by adding 4.5 mM CaCl2 to obtain a maximum signal and by adding medium with 20 mM EGTA/8 mM Tris yielding a minimum signal, both of these media containing 7.2 ␮M of the calcium ionophore ionomycin. For image quantification, background images were obtained in areas of the coverslips devoid of cells. Then, after background subtraction, each image obtained at 340 nm was divided by the corresponding

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image at 380 nm on a pixel-by-pixel basis and the mean pixel ratios of individual cells were obtained. [Ca2+ ]i was then calculated from these ratios using the formula given by Grynkiewicz et al. (1985) and a Kd value of 680 nM, which has been previously determined for our experimental set (Krumschnabel et al., 2001). 2.5. Detection of reactive oxygen species The production of ROS by goldfish hepatocytes was measured by use of the fluorescence indicator DCF-DA, as described for trout hepatocytes (Manzl et al., 2004) with slight modifications. For this purpose, 100 ␮l aliquots of a hepatocyte suspension (5 × 106 cells ml−1 ) were pipetted into the wells of a 96 well plate and were then mixed with 150 ␮l of saline containing Tris–HCl instead of HEPES and including DCF-DA (final concentration 3.6 ␮M). The fluorescence indicator diffuses into the cells where it is deacetylated to DCF, which reacts with intracellular ROS to form the fluorescent product 2 7 -dichlorofluoroscein. Fluorescence intensity was measured after 0, 5, 15, 30, 60 and 120 min of incubation using a Fluorescence Microplate-Reader (Molecular Devices) with excitation at 485 nm and emission set to 538 nm. Rates of ROS formation were presented by given the relative increase of DCF oxidation over the first 30 min after metal addition (ROS 0–30 min), so as to evaluate immediate effects of the various treatments applied. In order to investigate the effect of radical scavengers (200 ␮M DFA, 150 mM DMSO, 100 ␮M DTTC) and metabolic inhibitors (1 mM CN, 10 ␮M CCCP) on Cr(VI)-induced ROS formation, these were added to the cells immediately before the addition of Cr(VI) at time zero, an exception being DDTC, which was added 15 min before the metal. Following this incubation, extracellular DDTC was removed by briefly spinning the cells, aspiration of the supernatant, and resuspending the cells in fresh saline. Apart from DMSO, which was applied in pure form, all these substances were dissolved in water or DMSO and were added from concentrated stock solutions so as to keep dilution and the amount of DMSO added at a minimum. Furthermore, for evaluation of the effect of pre-treatment with Vitamin E the cells were cultured in modified L-15 medium with either 0.1% DMSO only (controls) or with 100 ␮M Vitamin E dissolved in DMSO. In this

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case hepatocytes were plated onto untreated coverslips to facilitate recovery of the cells on the next day. 2.6. Oxygen consumption The effect of Cr(VI) on oxygen consumption of goldfish hepatocytes was determined with a two chamber Cyclobiosis Oxygraph as described by Krumschnabel et al. (1994). Cells (5 × 106 hepatocytes ml−1 ) were injected into a 1500 ␮l measuring chamber containing standard incubation medium by use of a Hamilton syringe. When a constant baseline was observed Cr(VI), at 250 ␮M final concentration, was added to the suspension. Control cells were always measured in parallel to each metal exposure using the second measuring chamber and received an equal volume of distilled water. Cellular respiration was then determined over 30 min, and after termination of each experiment cells were recounted in a B¨urker–T¨urk hemocytometer. 2.7. Lysosomal membrane stability Lysosomal membrane stability was estimated from the capacity of the cells to retain neutral red. In short, cells attached to poly-l-lysine coated coverslips were loaded with neutral red (0.4% in standard saline) for 1 h and were then thoroughly washed with standard incubation medium before experimental exposure. The cells were mounted and examined on the microscopic set-up given above for [Ca2+ ]i measurements. Transmission light images were acquired every 2.5 min, including 5 min before and 60 min after Cr(VI) addition. Neutral red retention was calculated from the absorbency of stained sub regions of the cells and was expressed as percent of the mean absorbency before metal addition. 2.8. Statistics All data are presented as means ± S.E. of the number n independent preparations, where each preparation represents cells obtained from the livers of one or two fish (experiments with cell suspensions), or individual cells ([Ca2+ ]i measurements and lysosomal stability). In the latter case, at least three different cell cultures from three independent preparations were used. Differences between treatments were evaluated with either

analysis of variance (ANOVA) followed by Tukey’s post-hoc test or with Student’s t-test. These statistical tests were conducted on original data before conversion to percentage values where these are presented. A p-value < 0.05 was considered as significant.

3. Results As pointed out in the Section 1, the aim of the present study was to elucidate acute toxic effects of Cr(VI) and to identify mechanisms involved in Cr(VI) toxicity. Since preliminary experiments indicated that 250 ␮M Cr(VI) is the lowest concentration of the metal causing significant effects within the time frame investigated, this concentration of Cr(VI) was used throughout this study unless otherwise indicated. 3.1. Cell viability Cell viability determined by Trypan blue exclusion indicated that, after 30 min of exposure to 250 ␮M Cr(VI), a significant reduction from 94.0 ± 1.9% in controls to 88.3 ± 3.2% in Cr-treated cells was induced by the metal (n = 4; p < 0.05). Further exposure over 1 and 2 h caused only a slight further reduction of viability to 86.6 ± 2.1% and 86.2 ± 2.1%, respectively, and after 4 h of Cr-exposure viability still amounted to 83.9 ± 2.6%, which was not significantly different from the value after 30 min. In comparison, control cells maintained a viability of 93.6 ± 0.6% after 4 h of incubation (p < 0.05 compared to time-matched Crtreated cells). 3.2. Cytosolic free calcium Incubation of goldfish hepatocytes with 250 ␮M Cr(VI) caused no significant increase of [Ca2+ ]i within 60 min of incubation, the initial level of [Ca2+ ]i amounting to 64 ± 9 nM (n = 26 cells) and the value after 60 min to 67 ± 7 nM. As a positive control, indicating that these hepatocytes were in fact responsive to a physiological stimulus, we also treated hepatocytes with 1 ␮M epinephrine. In this case, the cells showed a clear rise of [Ca2+ ]i up to 800 nM with fluctuations of [Ca2+ ]i , which we have previously found to be a typical response of these cells to epinephrine (Krumschnabel et al., 2001; Manzl et al., 2002).

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3.3. Formation of reactive oxygen species Oxidation of DCF, serving as a measure of ROS formation, was used to determine the hypothetical role of radical stress in Cr(VI) toxicity. Since previous studies have found that the induction of ROS formation by a redox active metal may occur well before other toxic effects can be detected (Pourahmad et al., 2003; Manzl et al., 2004), we regard this parameter a rather sensitive indicator of metal toxicity. Therefore, we established a dose-response curve of ROS formation for Cr(VI), the result of which is depicted in Fig. 1. As

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can be seen, there was no increase of ROS formation detectable with 1 and 10 ␮M Cr(VI), and a significant 50–100% enhancement above control rates was noted with 100 ␮M. With 250 ␮M Cr(VI), a much more pronounced increase of cellular ROS formation was induced, which amounted to more than 500% of control rates during the first 30 min of incubation. Thereafter, the rate of DCF oxidation increase slowed down. Cellular production of ROS was further studied with several compounds potentially interfering with the effect of Cr(VI) (Susa et al., 1996, 1997, 1998). As shown in Table 1, the ferric ion chelator DFA (200 ␮M final concentration) caused a significant reduction of basal ROS formation, i.e. in the absence of Cr(VI), and also partially suppressed Cr-induced ROS production. The hydroxyl radical scavenger DMSO (150 mM) caused a slight increase of ROS formation in the absence of Cr(VI). In contrast, with Cr(VI) present, it tended to reduce ROS formation, but this was not quite significant. In addition, we investigated the effect of a pre-treatment of hepatocytes with the antioxidant Vitamin E for 24 h (100 ␮M), or with the metal chelator Table 1 Formation of reactive oxygen species (ROS) of goldfish hepatocytes in the absence and presence of 250 ␮M Cr(VI) and of various radical scavengers or metabolic inhibitors

Fig. 1. Formation of reactive oxygen species (ROS) by goldfish hepatocytes, as estimated from the oxidation of DCF, exposed to different concentrations of Cr(VI). Upper panel: time course of DCF oxidation over 2 h of incubation under the conditions indicated. Lower panel: rate of DCF oxidation over the first 30 min of incubation, expressed as percent of the rate determined in control cells, i.e. in the absence of Cr(VI). Data are expressed as means ± S.E. from 5–6 cell preparations. * p < 0.05 compared to untreated controls.

Treatment

ROS (0–30 min)

Cr(VI) +DFA +DMSO Cr(VI) +Vit E + DDTC Cr(VI) +CN +CCCP Control +DFA +DMSO +Vit E + DDTC +CN +CCCP

430 ± 48 (5) 364 ± 27a (5) 373 ± 71 (5) 612 ± 48 (9) 625 ± 95 (7) 1027 ± 284a (6) 523 ± 50 (10) 302 ± 32a (10) 417 ± 30 (8) 100 60 ±7b (5) 155 ± 12b (5) 111 ± 6 (7) 242 ± 65b (6) 76 ± 6b (10) 96 ± 5 (8)

ROS formation was estimated from the rate of DCF oxidation over the first 30 min after metal addition (ROS, 0–30 min). Data are expressed as percentage of the rate determined in control cells, i.e. in the absence of Cr(VI) and are means ± S.E. of the number of experiments given in parentheses. a p < 0.05 vs. Cr(VI). b p < 0.05 vs. controls.

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DDTC for 15 min (1 mM), since these treatments have been previously found to reduce oxidative stress in rat hepatocytes exposed to Cr(VI) (Susa et al., 1996, 1998). However, these treatments neither reduced basal nor Cr-induced ROS-formation, but DDTC even significantly enhanced ROS formation relative to that seen in its absence. Finally, we also tested the effect of agents interfering with mitochondrial function, so as to assess the hypothetical role of mitochondrially generated radicals. In fact, we found that the cytochrome c oxidase inhibitor CN significantly inhibited the formation of ROS, both in the presence and absence of Cr(VI). In contrast, the mitochondrial uncoupling agent CCCP was without any effect on basal ROS production and on Cr(VI)-stimulated radical formation. 3.4. Oxygen consumption (VO2 ) In the absence of Cr(VI), cellular VO2 amounted to 0.592 ± 0.093 nmol O2 10−6 cells min−1 (n = 5) and remained more or less constant during the 30 min measuring period (Fig. 2). In contrast, within one min of exposure to Cr(VI), goldfish hepatocytes showed a substantial increase of their respiratory rates to 122 ± 5% of the initial level, and VO2 remained elevated during the next 5 min. Subsequently, VO2 dropped back

Fig. 2. Rates of oxygen consumption (VO2 ) of goldfish hepatocytes during 30 min of incubation following the addition of 250 ␮M Cr(VI) or the solvent, i.e. aqua dest. Results are expressed as percent of initial respiratory rate, determined before addition of the metal. Values are means ± S.E. of five independent preparations. (*) p < 0.05 compared to initial rate.

Fig. 3. Lysosomal membrane stability, as estimated from neutral red retention, in goldfish hepatocytes incubated for 60 min with or without 250 ␮M Cr(VI) or with 100 ␮M CuCl2 . Results are expressed as percent of the mean absorbency before addition of the respective metal. Data are means ±S.E. of 42 (Cr) and 12 (Cu) individual cells from three to four independent cultures each.

to 107 ± 4% and 84 ± 6% of the control level after 15 and 30 min, respectively. 3.5. Lysosomal membrane stability During 1 h of incubation with 0.4% neutral red goldfish hepatocytes accumulated the dye in the intracellular space and displayed densely stained lysosomal vesicles (not shown). Following the addition of Cr(VI), the cells did not appear to release the stain within the 60 min investigated, suggesting that no lysosomal leakage was induced by the metal (Fig. 3). In order to check the validity of the method used for the assessment of lysosomal membrane stability, we conducted a series of experiments with 100 ␮M copper, which we recently found to be highly toxic in hepatocytes from another fish species (Manzl et al., 2003, 2004). Indeed, as depicted in Fig. 3, with 100 ␮M Cu present the cells started to release the stain within about 30 min of exposure, and numerous cells showed membrane blebbing and ultimately burst (not shown).

4. Discussion In line with several studies on mammalian cell models, our present study showed that acute exposure

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of goldfish hepatocytes to Cr(VI) significantly reduced cell viability and stimulated an increased production of ROS. Concomitant with these effects we observed a transient elevation of cellular oxygen consumption, whereas Ca2+ homeostasis and lysosomal membrane stability remained unaffected by the metal. In comparison to other metals a rather high concentration of Cr(VI), i.e. 250 ␮M, was required in order to induce cytotoxic effects. This, however, agrees with studies on rat hepatocytes, where 250 ␮M Cr(VI) had to be applied to induce significant cell death after 8 h of incubation (Susa et al., 1998), and an LC50 of 1 mM, as determined for a 3 h exposure period (Pourahmad and O’Brien, 2001). Furthermore, our finding that 250 ␮M Cr(VI) was also the lowest concentration evoking a pronounced rise of ROS formation suggests that radical stress is closely linked to the toxic action of Cr(VI). In agreement with this, a number of investigations has shown that measures intervening with ROS formation confer significant protection against Cr(VI) toxicity (Susa et al., 1996, 1997; Pourahmad et al., 2003). Specifically, it was shown that e.g. Vitamin E (Susa et al., 1996) or DDTC (Susa et al., 1998) caused a drastic reduction of lipid peroxidation caused by Cr(VI), which was estimated from the formation of malondialdehyde. In goldfish hepatocytes, we found that neither the hydroxyl radical scavenger DMSO nor Vitamin E pre-treatment reduced Cr(VI) induced ROS formation. This may indicate that the origin of radicals is neither cytosolic, where DMSO would interfere with ROS formation, nor are these radicals formed at the cell membrane, where Vitamin E is primarily located (Susa et al., 1996). This does, however, not preclude that these agents diminished lipid peroxidation stimulated by Cr(VI), which may not be detectable by measuring DCF oxidation. The same may hold for the metal chelator DTTC, although this compound even enhanced ROS formation. While this observation remains unexplained at the moment, we nevertheless could identify at least two significant sources contributing to enhanced formation of ROS. On the one hand, the partial inhibition of ROS formation by the ferric ion chelator DFA indicates an extra-mitochondrial source. As has been suggested by Pourahmad et al. (2003), these ROS may originate from the lysosomal compartment, which represents a major ¨ part of the cellular pool of reactive iron (Ollinger and Brunk, 1995; Yu et al., 2003). There, free Fe2+ may

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undergo redox cycling to form ROS from hydrogen peroxide, which has been previously formed by redox cycling of Cr(VI) in the cytoplasm. In line with this, it has been shown that DFA is mainly taken up by endocytosis and thus transported to the acidic vacuoles ¨ such as the lysosomes (Lloyd et al., 1991; Ollinger and Brunk, 1995). Interestingly, if the lysosomes were indeed an important source of ROS in goldfish hepatocytes, our experiments indicate that disruption of lysosomal membrane stability is not a prerequisite for enhanced ROS formation in these organelles, as in the hepatocytes neutral red retention was clearly preserved in the presence of Cr(VI). On the other hand, our data suggest that mitochondria contributed to ROS production in the goldfish cells. Addition of CN, which causes near complete inhibition of oxygen consumption in these cells (Krumschnabel et al., 1994), significantly suppressed ROS formation even in the absence of Cr(VI). This is in line with the general notion that mitochondria represent an important source of oxygen radicals of basal metabolism (Halliwell and Gutteridge, 1999). Similarly, CN significantly diminished ROS formation in the presence of Cr(VI), indicating that the metal also acts on the mitochondria. This is underlined by the finding that oxygen consumption was significantly increased after Cr(VI) addition. Interestingly, this is at variance with several reports showing a decrease of mitochondrial respiration in the presence of Cr(VI), both in intact cells as well as in isolated mitochondria (Lazzarini et al., 1985; Ryberg and Alexander, 1990). However, in isolated rat mitochondria Fernandes et al. (2002) observed a pronounced increase of state 4 respiration at a comparable concentration of Cr(VI), indicating either enhanced proton leak across the mitochondrial membrane or increased ATPase activity. In the intact goldfish hepatocytes enhanced oxygen turnover may thus represent either respiration coupled to ATP production or uncoupled respiration. Given that in various cells Cr(VI) caused a strong decrease of cellular ATP levels (Bianchi et al., 1982; Lazzarini et al., 1985), and the large reduction of mitochondrial membrane potential reported for rat hepatocytes exposed to Cr(VI) (Pourahmad et al., 2001), the latter appears more likely. In agreement with this, the addition of the mitochondrial uncoupling agent CCCP did not inhibit the formation of ROS. A similar lack of CCCP inhibiting ROS formation has been previously observed in rat

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hepatocytes exposed to Cr(VI) (Pourahmad et al., 2003). The precise mechanism underlying the increase of presumably uncoupled respiration remains unexplained at the moment. Our initial assumption was that a rise of [Ca2+ ]i may have stimulated mitochondrial activity, analogous to the Ca2+ -dependent elevation of oxygen consumption previously observed in trout hepatocytes exposed to copper (Manzl et al., 2003). This, however, appears to be invalid, as we could not detect any increase of [Ca2+ ]i in goldfish hepatocytes exposed to Cr(VI), whereas they were clearly responsive to a physiological stimulus such as epinephrine. Data available for other cells indicate that Cr(VI) exposure may induce a rise of Ca2+ (Tarnok et al., 2001; Hayashi et al., 2004), but also a decrease of Ca2+ influx (Nasu et al., 1993; Liu and Lin, 1997), which may lead to a decrease of [Ca2+ ]i . The insensitivity of goldfish hepatocytes in this regard may reflect the generally outstanding capability of these cells to maintain ion homeostasis under a variety of challenging conditions (Krumschnabel et al., 1996; Espelt et al., 2003). In summary, we found that Cr(VI) is acutely toxic to goldfish hepatocytes, and this is associated with the induction of radical formation. The radicals may partly originate from the lysosomes, which, nevertheless remain intact in the presence of Cr(VI). An additional source of ROS are the mitochondria, where a Ca2+ -independent, and presumably uncoupled increase of respiration is triggered by the metal.

Acknowledgements This study was supported by the Fonds zur F¨orderung der wissenschaftlichen Forschung in ¨ Osterreich, project no. P16154-B06. M. Nawaz is ¨ recipient of a grant of the OAD-Pakistan scholarship programme. We thank Prof. Bernd Pelster for critical reading of the manuscript.

References Bagchi, D., Stohs, S.J., Downs, B.W., Bagchi, M., Preuss, H.G., 2002. Cytotoxicity and oxidative mechanisms of different forms of chromium. Toxicology 180, 5–22.

Bianchi, V., Debetto, P., Zantedeschi, A., Levis, A.G., 1982. Effects of hexavalent chromium on the adenylate pool of hamster fibrolasts. Toxicology 25, 19–30. Birnbaum, M.J., Schultz, J., Fain, J.N., 1976. Hormone-stimulated glycogenolysis in isolated goldfish hepatocytes. Am. J. Physiol. 231, 191–197. Espelt, M.V., Mut, P.N., Amodeo, G., Krumschnabel, G., Schwarzbaum, P.J., 2003. Volumetric and ionic responses of goldfish hepatocytes to anisotonic exposure and energetic limitation. J. Exp. Biol. 206, 513–522. Fernandes, M.A., Santos, M.S., Alpoim, M.C., Madeira, V.M., Vicente, J.A., 2002. Chromium(VI) interaction with plant and animal mitochondrial bioenergetics: a comparative study. J. Biochem. Mol. Toxicol. 16, 53–63. Grynkiewicz, G., Poenie, M., Tsien, R.Y., 1985. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440–3450. Halliwell, B., Gutteridge, J.M.C., 1999. Free Radicals In Biology and Medicine. Oxford University Press, Oxford. Hayashi, Y., Kondo, T., Zhao, Q.-L., Ogawa, R., Cui, Z.-G., Feril Jr., L.B., Teranishi, H., Kasuya, M., 2004. Signal transduction of p53-independent apoptotic pathway induced by hexavalent chromium in U973 cells. Toxicol. Appl. Pharmacol., in press. Krumschnabel, G., Schwarzbaum, P.J., Wieser, W., 1994. Coupling of energy supply and energy demand in isolated goldfish hepatocytes. Physiol. Zool. 67, 438–448. Krumschnabel, G., Biasi, C., Schwarzbaum, P.J., Wieser, W., 1996. Membrane-metabolic coupling and ion homeostasis in anoxiatolerant and anoxia-intolerant hepatocytes. Am. J. Physiol. 270, R614–R620. Krumschnabel, G., Manzl, C., Schwarzbaum, P.J., 2001. Metabolic responses to epinephrine stimulation in goldfish hepatocytes: evidence for the presence of alpha-adrenoceptors. Gen. Compd. Endocrinol. 121, 205–213. Lazzarini, A., Luciani, S., Beltrame, M., Arslan, P., 1985. Effects of chromium(VI) and chromium(III) on energy charge and oxygen consumption in rat thymocytes. Chem. Biol. Interact. 53, 273–281. Liu, P.S., Lin, M.K., 1997. Biphasic effects of chromium compounds on catecholamine secretion from bovine adrenal medullary cells. Toxicology 117, 45–53. Liu, K.J., Shi, X., 2001. In vivo reduction of chromium (VI) and its related free radical generation. Mol. Cell Biochem. 222, 41–47. Lloyd, J.B., Cable, H., Rice-Evans, C., 1991. Evidence that desferrioxamine cannot enter cells by passive diffusion. Biochem. Pharmacol. 41, 1361–1363. Manzl, C., Schubert, M., Schwarzbaum, P.J., Krumschnabel, G., 2002. Effects of chemical anoxia on adrenergic responses of goldfish hepatocytes and the contribution of alpha- and betaadrenoceptors. J. Exp. Zool. 292, 468–476. Manzl, C., Ebner, H., Kock, G., Dallinger, R., Krumschnabel, G., 2003. Copper, but not cadmium, is acutely toxic for trout hepatocytes: short-term effects on energetics and ion homeostasis. Toxicol. Appl. Pharmacol. 191, 235–244. Manzl, C., Enrich, J., Ebner, H., Dallinger, R., Krumschnabel, G., 2004. Copper-induced formation of reactive oxygen species

G. Krumschnabel, M. Nawaz / Aquatic Toxicology 70 (2004) 159–167 causes cell death and disruption of calcium homeostasis in trout hepatocytes. Toxicology 196, 57–64. Nasu, T., Ooyama, I., Shibata, H., 1993. Inhibitory effects of hexavalent chromium ions on the contraction in ileal longitudinal smooth muscle of guinea-pig. Comp. Biochem. Physiol. C 104, 97–102. Nicotera, P., Bellomo, G., Orrenius, S., 1992. Calcium-mediated mechanisms in chemically induced cell death. Annu. Rev. Pharmacol. Toxicol. 32, 449–470. ¨ Ollinger, K., Brunk, U.T., 1995. Cellular injury induced by oxidative stress is mediated through lysosomal damage. Free Radic. Biol. Med. 19, 565–574. Pourahmad, J., O’Brien, P.J., 2001. Biological reactive intermediates that mediate chromium (VI) toxicity. Adv. Exp. Med. Biol. 500, 203–207. Pourahmad, J., Mihajlovic, A., O’Brien, P.J., 2001. Hepatocyte lysis induced by environmental metal toxins may involve apoptotic death signals initiated by mitochondrial injury. Adv. Exp. Med. Biol. 500, 249–252. Pourahmad, J., O’Brien, P.J., Jokar, F., Daraei, B., 2003. Carcinogenic metal induced sites of reactive oxygen species formation in hepatocytes. Toxicol. In Vitro 17, 803–810. Rissanen, E.H., Krumschnabel, G., Nikinmaa, M., 2002. Dehydroabietic acid, a major component of wood industry effluents, interferes with cellular energetics in rainbow trout hepatocytes. Aquat. Toxicol. 62, 45–53. Ryberg, D., Alexander, J., 1990. Mechanisms of chromium toxicity in mitochondria. Chem. Biol. Interact. 75, 141– 151. Shi, X.L., Dalal, N.S., 1990. Evidence for a Fenton-type mechanism for the generation of OH• radicals in the reduction of Cr(VI) in cellular media. Arch. Biochem. Biophys. 281, 90– 95. Stift, A., Friedl, J., Langle, F., Berlakovich, G., Steininger, R., M¨uhlbacher, F., 2000. Successfull treatment of a patient suffering from severe acute potassium dichromate poisoning with liver transplantation. Transplantation 69, 2454–2455.

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Sugiyama, M., Tsuzuki, K., Hidaka, T., Ogura, R., Yamamoto, M., 1991. Reduction of chromium(VI) in Chinese hamster V-79 cells. Biol. Trace Elem. Res. 30, 1–8. Susa, N., Ueno, S., Furukawa, Y., Sugiyama, M., 1996. Protective effect of Vitamin E on chromium (VI)-induced cytotoxicity and lipid peroxidation in primary cultures of rat hepatocytes. Arch. Toxicol. 71, 20–24. Susa, N., Ueno, S., Furukawa, Y., Sugiyama, M., 1997. Protective effect of deferoxamine on chromium (VI)-induced DNA singlestrand breaks, cytotoxicity, and lipid peroxidation in primary cultures of rat hepatocytes. Arch. Toxicol. 71, 345–350. Susa, N., Ueno, S., Furukawa, Y., 1998. Protective effect of diethyldithiocarbamate pretreatment on chromium (VI)-induced cytotoxicity and lipid peroxidation in primary cultures of rat hepatocytes. J. Vet. Med. Sci. 60, 71–76. Tan, S., Sagara, Y., Liu, Y., Maher, P., Schubert, D., 1998. The regulation of reactive oxygen species production during programmed cell death. J. Cell Biol. 141, 1423–1432. Tarnok, A., Dorger, M., Berg, I., Gercken, G., Schluter, T., 2001. Rapid screening of possible cytotoxic effects of particulate air pollutants by measurement of changes in cytoplasmic free calcium, cytosolic pH, and plasma membrane potential in alveolar macrophages by flow cytometry. Cytometry 43, 204–210. Thaker, J., Chhaya, J., Nuzhat, S., Mittal, R., Mansuri, A.P., Kundu, R., 1996. Effects of chromium(VI) on some ion-dependent ATPases in gills, kidney and intestine of a coastal teleost Periophthalmus dipes. Toxicology 112, 237–244. van der Putte, I., Laurier, M.B.H.M., van Eijk, G.J.M., 1982. Respiration and osmoregulation in rainbow trout (Salmo gairdneri) exposed to hexavalent chromium at different pH values. Aquat. Toxicol. 2, 99–112. Vutukuru, S.S., 2003. Chromium induced alterations in some biochemical profiles of the Indian major carp, Labeo rohita (Hamilton). Bull. Environ. Contam. Toxicol. 70, 118–123. Yu, Z., Persson, H.L., Eaton, J.W., Brunk, U.T., 2003. Intralysosomal iron: a major determinant of oxidant-induced cell death. Free Radic. Biol. Med. 15, 1243–1252.