Re-evaluation of acute neurotoxic effects of Cd2+ on mesencephalic trigeminal neurons of the adult rat

Brain Research 892 (2001) 102–110 www.elsevier.com / locate / bres

Research report

Re-evaluation of acute neurotoxic effects of Cd 21 on mesencephalic trigeminal neurons of the adult rat Shigeru Yoshida* Department of Physiology, Fukui Medical School, Matsuoka, Fukui 910 -1193, Japan Accepted 14 November 2000

Abstract The mechanism of Cd 21 neurotoxicity, which is considered to be secondary to changes in blood vessels, was re-evaluated in dissociated mesencephalic trigeminal (Me5) neurons of the adult rat. Cd 21 induced morphological changes in Me5 neurons at 0.1 and 1 mM but not at 0.01 mM. The changes appeared predominantly in the cytoplasm: destruction of the cytoplasmic organelles, swelling and vacuolization of the cell body, and finally resulted in cell lysis. These observations indicate necrosis rather than apoptosis, and no sign of degraded nuclear DNA, characteristic to apoptosis, was detected by the TUNEL technique. Using a Ca 21 -sensitive dye Indo-1, Cd 21 was found to elevate the intracellular Ca 21 concentration [Ca 21 ] i (both in the cytoplasm and the nucleus). Both the elevation in [Ca 21 ] i and the morphological alteration were inhibited either by removing Ca 21 from the bathing medium or by the application of BAPTA /AM (10 mM), a membrane-permeable intracellular Ca 21 chelator. Furthermore, neither morphological changes nor elevation in [Ca 21 ] i by Cd 21 occurred in the presence of Zn 21 . It is concluded that (1) Cd 21 can directly affect nerve cells, (2) toxicity of Cd 21 on Me5 neurons is mediated by continuous elevation in [Ca 21 ] i , (3) Cd 21 induces necrotic cell death, and (4) Cd 21 neurotoxicity can be antagonized by Zn 21 .  2001 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Neurotoxicity Keywords: Mesencephalic trigeminal neuron; Ca 21 concentration; Cd 21 ; Neurotoxicity; Necrosis

1. Introduction Adverse health effects of cadmium (Cd 21 ) are of particular concern because this metal can be easily taken into human bodies in various occupational or environmental settings or through contaminated substances (air, foods, water, tobacco etc.) [6,7,18,19,21,24,41]. Because of its long biological half-life of 15–20 years in humans, chronic exposure to Cd 21 not only produces multiorgan damage (especially in the kidney, lung and bone), but also acts as a carcinogen and as a teratogen in humans and experimental animals [5,17,19,21,26,37,38,41]. The nervous system is one of the targets for the Cd 21 toxicity [6,16,29]. However, the direct adverse effect of Cd 21 on neurons has been a matter of controversy, because *Present address: Department of Physiology, Nagasaki University School of Medicine, Nagasaki 852-8523, Japan. Fax: 181-95-849-7036. E-mail address: [email protected] (S. Yoshida).

a number of studies suggested that the initial effect of Cd 21 administration was on cerebral blood vessels, especially endothelial cell damage with hemorrhages and interstitial edema, and that the necrotic changes occurred in nerve cells secondarily, mainly by anoxia and edema [3,11,12,25,28,40]. The aim of the present work is to re-evaluate the direct effect of acute Cd 21 application on nerve cells using dissociated mesencephalic trigeminal (Me5) neurons to eliminate the vascular component from the study.

2. Materials and methods

2.1. Preparation of Me5 neurons Mesencephalic trigeminal (Me5) neurons were isolated from adult rats (ages between 8 and 12 weeks, both sexes) as described previously [44]. Briefly, a midbrain tissue

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block was obtained from a rat under ether anesthesia, and it was chopped into brain slices of 500 mM thickness. The Me5 nucleus was trimmed off from the slices under a stereomicroscope, and Me5 neurons were isolated with 5 mg / ml trypsin (Type XI, Sigma).

2.2. Ca 21 measurements in Me5 neurons Dissociated Me5 neurons were plated on glass coverslips, and they were loaded with 10 mM Indo-1 acetoxymethyl ester (Indo 1 /AM; Dojindo Laboratories, Kumamoto, Japan) plus 0.02% pluronic F-127 detergent (Sigma) for 20–30 min at 348C. Both Indo-1 and pluronic F-127 were firstly dissolved in dimethyl sulphoxide (DMSO, Sigma) before dilution with the extracellular standard solution. An experimental chamber with a glass coverslip bottom was placed on the stage of the laser-scanning confocal microscope (model LSM-GB 200 UV, Olympus) for Ca 21 measurement. Indo-1 fluorescence was induced by epi-illumination with 35065 nm light, and Indo-1 emission was obtained at 395–415 and 470–490 nm for estimation of [Ca 21 ]. Collected data were analyzed by an IBM AT-compatible computer using the analysis software (LSM GB200Ca, Olympus). Calculation of [Ca 21 ] was performed using the method proposed by Grynkiewicz et al. [14].

2.3. TUNEL reaction In order to detect apoptotic changes of cells, the TUNEL (TdT-mediated deoxyuridine triphosphate–biotin nick endlabeling) technique was applied to Me5 neurons. Neurons were fixed with 4% formalin for 20 min and stained using a commercially available TUNEL-kit (in situ apoptosis detection kit MK500, Takara Biomedicals, Shiga Prefecture, Japan). TUNEL-labeled cells were observed by the same laser-scanning confocal microscope (model LSM-GB 200 UV, Olympus) used for the Ca 21 measurements.

2.4. Solutions The composition of the standard extracellular solution used for isolation and staining of Me5 neurons and for experiments was (mM): 145 NaCl, 5 KCl, 2.5 CaCl 2 , 1 21 MgCl 2 , 10 HEPES and 10 glucose (pH 7.4). The Ca free solution contained (mM): 145 NaCl, 5 KCl, 3.5 MgCl 2 , 2 EGTA, 10 HEPES and 10 glucose (pH 7.4). Solutions were applied to Me5 neurons by perfusion at the speed of 0.5–1 ml / min using a polyethylene tube (500 mm inner diameter) placed approximately 2 mm apart from neurons. A membrane-permeable intracellular Ca 21 chelator BAPTA /AM, an acetoxymethyl ester of BAPTA (bis-(o-aminophenoxy)-ethane-N,N,N9,N9-tetraacetic acid), was purchased from Calbiochem (La Jolla, USA). BAPTA /AM was firstly dissolved in DMSO and applied to

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the bathing medium at the concentration of 0.1% (v / v). The vehicle DMSO showed no effect on its own at this concentration. Ethylene glycol bis-(b-aminoethylether)N,N9-tetraacetic acid (EGTA) was obtained from Fluka and used as an extracellular Ca 21 chelator. Actinomycin D was obtained from Sigma. All experiments were carried out at room temperature (21–238C).

3. Results

3.1. Morphological changes induced by Cd 21 Morphological changes in nerve cells are a good indication of neurotoxicity of metals [12,40]. In the present work, rapid alterations in morphology were evoked in Me5 neurons by the application of Cd 21 by perfusion. A representative example of a Me5 neuron which was exposed to 1 mM CdCl 2 is shown in Fig. 1 (n515). The photos illustrate time-dependent granulation of the cytoplasm, swelling of the cell body, and cytoplasmic vacuolization (at the 3 o’clock position). These cytoplasmic changes finally lead this neuron to cell lysis and the release of cellular contents into the bathing medium. The nucleus became pyknotic. These morphological changes were dependent on the concentration of applied Cd 21 . No apparent changes were detected at 0.01 mM Cd 21 within 2 or 3 h (n510). However, 0.1 mM Cd 21 induced similar changes to those illustrated in Fig. 1 (n57). These morphological changes induced by 0.1 and 1 mM Cd 21 were hard to reverse once they started even by washing out Cd 21 from the bathing medium for 1–3 h, suggesting that the effect of Cd 21 is irreversible.

3.2. Morphological changes induced by Cd 21 indicate necrosis rather than apoptosis Neuronal cell death is classified into two major entities, necrosis and apoptosis. Characteristic morphological changes of apoptosis are shrinkage of the cell body, nuclear pyknosis (chromatin condensation), and nuclear fragmentation (apoptotic body) [27]. On the contrary, necrotic changes predominantly appear in the cytoplasm: destruction of cytoplasmic organelles, especially mitochondria, swelling of the cell body, disruption of the cell membrane leading to release of the cytoplasm [3,27]. In this context, the Cd 21 -induced changes observed in Me5 neurons (Fig. 1) indicate necrosis rather than apoptosis. In order to further assess the necrotic cell death, the TUNEL technique was applied to Cd 21 -treated Me5 neurons. This technique is conventionally used to detect apoptotic nuclear morphology (nuclear DNA fragmentation). Fig. 2A illustrates a control Me5 neuron which was not exposed to Cd 21 . A bit-image of a Me5 neuron is shown in A1 and its fluorescence micrograph in A2. Control neurons exhibited no or very weak fluorescence images as in A2 (n522).

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Fig. 1. Photomicrographs of morphological changes induced by Cd 21 in a mesencephalic trigeminal (Me5) neuron which was dissociated from an adult rat. A trace of truncated axon is seen as a curtailed lump at the 10 o’clock position. Time indicates the time lapse since 1 mM Cd 21 was introduced into the experimental chamber by perfusion. Scale bar of 20 mm applies to all images.

Although slightly clearer fluorescence images were obtained from Me5 neurons exposed to 1 mM Cd 21 for 1–2 h (B2), compared to controls, no apparent sign of apoptotic nuclear morphology, such as clumped or segmented nuclei, was observed (n516). For comparison, a TUNEL-positive fluorescence micrograph of HL-60 human promyelocytic leukemic cells is displayed in Fig. 2C. The HL-60 cells were treated with 1 mg / ml actinomycin D for 3 h at 378C and fixed with 4% formalin. Actinomycin D is an inhibitor of DNA-primed RNA polymerase and is known as a potent inducer of apoptosis [20]. Note the prominent segmented nuclei characteristic of apoptotic nuclear morphology (C2).

3.3. Intracellular Ca 21 elevation evoked by Cd 21 Divalent cations including Cd 21 (Cd 21 , Ni 21 , Zn 21 , 21 21 21 21 Co , Mn , Cr ) are reported to elevate [Ca ] i [23]

and a sustained increase in [Ca 21 ] i is believed to be one of the major causes of cell death [27]. Therefore, the intracellular Ca 21 concentration was measured using a Ca 21 sensitive dye Indo-1. A representative example of the effect of Cd 21 is displayed in Fig. 3. Pseudo-color bitimages of the same Me5 neuron as shown in Fig. 1, exhibit dynamic changes in [Ca 21 ] i monitored with a laser-scanning confocal microscope. Stronger reddening of an image indicates a higher concentration of Ca 21 . The images illustrate that [Ca 21 ] i gradually increased with time when the Me5 neuron was continuously exposed to Cd 21 . Since the nucleus could be distinguished from the cytoplasm (see Figs. 1 and 3), both the concentrations of Ca 21 in the cytoplasm ([Ca 21 ] c ) and in the nucleus ([Ca 21 ] n ) were measured in the present work. Time-lapse changes in [Ca 21 ] c (solid curve) and [Ca 21 ] n (dotted curve) are shown in Fig. 4. Photomicrographs of the neuron (shown in Fig.

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Fig. 2. Detection of apoptotic signs by the TUNEL technique. A: Control Me5 neuron. B: TUNEL-negative Me5 neuron treated with 1 mM Cd 21 for 1 h. C: TUNEL-positive segmented nuclei (C2) observed in HL-60 human promyelocytic leukemic cells which were treated with 1 mg / ml actinomycin D for 3 h. A1, B1, and C1 are photomicrographs and A2, B2, and C2 are their fluorescence micrographs. Scale bar shown applies to all images.

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Fig. 3. Pseudo-color images of the Me5 neuron showing the intracellular concentration of Ca 21 . Time indicates the time lapse since 1 mM Cd 21 was applied to the neuron by perfusion. Reddening of an image indicates that the Ca 21 concentration is elevated. Scale bar shown applies to all images.

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Fig. 4. Effect of Cd 21 on the Ca 21 concentration of the cytoplasm (solid curve) and the nucleus (dotted curve). Horizontal bar marked 1 mM Cd 21 shows when the Cd 21 was added to the perfusate. Data were collected every 6 s.

1) were taken during the gaps in the Ca 21 measurement. The graph shows that both [Ca 21 ] c and [Ca 21 ] n increased and reached a steady level when the neuron was continuously exposed to Cd 21 . The rise in [Ca 21 ] n was slightly larger than the rise in [Ca 21 ] c in this case, but this was not significant (n515). It is noteworthy that no sign of recovery in [Ca 21 ] i was observed even when Cd 21 was removed from the bathing medium (Fig. 4), indicating the effect of Cd 21 was irreversible. It is also to be noted that [Ca 21 ] i did not increase instantly but with some delay in time. This delay cannot be explained by the perfusion system used in the present work, because the tip of the perfusion tube was placed close to neurons (see Methods) and the time spent switching solutions was measured and compensated for. The elevation in [Ca 21 ] i with time was plotted for the different concentrations of applied Cd 21 (Fig. 5; n57). Since[Ca 21 ] c and [Ca 21 ] n were increased by Cd 21 concomitantly, only [Ca 21 ] c is shown in this graph with the standard deviation (S.D.). These results clearly show the dose-dependent nature of the Cd 21 response.

Fig. 5. Dose-dependent effect of Cd 21 on the cytoplasmic Ca 21 concentration of Me5 neurons (n57). Cd 21 was applied to neurons at time zero, and the concentration of Cd 21 is indicated by open circles (0.01 mM), filled circles (0.1 mM) and open triangles (1 mM). Bars show standard deviation (S.D.).

[Ca 21 ] i . For instance, Fig. 6 shows the concentration of cytoplasmic and nuclear Ca 21 before (0 min) and at 30 and 21 60 min after the extracellular application of 1 mM Cd (n58). It is to be noted that no apparent morphological changes were induced by Cd 21 in these BAPTA /AMpretreated Me5 neurons. The data indicate that a continu-

3.4. Suppression of the Cd 21 response by chelating intracellular Ca 21 In order to examine whether the sustained elevation of the cytoplasmic Ca 21 is responsible for the necrotic changes in Me5 neurons, the intracellular Ca 21 concentration was lowered by a chelator BAPTA. In the present study, an acetoxymethyl ester of BAPTA (BAPTA /AM) was used. When BAPTA /AM penetrates the cell membrane, it becomes membrane-impermeant BAPTA by being cleaved by cytoplasmic esterases. Me5 neurons were pretreated with 10 mM BAPTA /AM in the standard external solution for 2 h to allow a sufficient amount of BAPTA to be locked within neurons [36,45]. When BAPTA /AM-pretreated Me5 neurons were challenged by 0.1 or 1 mM Cd 21 , no significant changes were detected in

Fig. 6. Effect of preincubation with 10 mM BAPTA /AM, a membranepermeant intracellular Ca 21 chelator, for 2 h on the response to Cd 21 . The Ca 21 concentration of the cytoplasm (open circles) and the nucleus (filled circles) are plotted against time (n58). Cd 21 (1 mM) was applied to the neurons at time zero. Bars indicate S.D.

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ous elevation of the intracellular Ca 21 is responsible for the necrotic changes seen in nerve cells.

3.5. Involvement of extracellular Ca 21 in the Cd 21 response Two sources can provide Ca 21 when its intracellular concentration becomes elevated, entry of Ca 21 from outside the cell and release of Ca 21 from the intracellular stores [4,45]. In the case of Me5 neurons, Ca 21 entry seems to be contributing considerably to the Cd 21 response. When perfusate was switched from the standard extracellular solution to Ca 21 -free (containing 2 mM EGTA) solution, no significant elevation in [Ca 21 ] i was observed although the baseline oscillation became slightly larger (Fig. 7; n58). Under this Ca 21 -free (EGTA) condition, the application of Cd 21 did not induce any appreciable change in [Ca 21 ] i . The Ca 21 -free (EGTA) solution was made by replacing Ca 21 with an equal amount of Mg 21 , and by adding 2 mM EGTA to the solution, to chelate any residual trace of extracellular Ca 21 . This solution prevents the entry of Ca 21 from outside the neuron since the estimated concentration of extracellular Ca 21 was less than 0.1 nM (using the equation developed by Fabiato and Fabiato [10]). Indeed as shown in Fig. 7, reintroduction of standard extracellular solution, starting at the end of the horizontal bar marked Ca 21 -free (EGTA), restored the action of Cd 21 and elevated both cytoplasmic and nuclear Ca 21 concentrations. This indicates that the Cd 21 response is dependent on the entry of Ca 21 from outside the cell (n58).

3.6. Antagonistic actions of Zn 21 against Cd 21 It has been reported that Zn 21 protects against Cd 21 toxicity in various tissues [2,15,32] including peripheral

Fig. 7. Inhibition of the Cd 21 response under Ca 21 -free conditions. The horizontal bar marked Ca 21 -free (EGTA) indicates that the perfusate was switched to that solution from the extracellular standard solution, and the bar marked 1 mM Cd 21 shows that Cd 21 was added to the perfusate at the indicated concentration. Image data were collected every 5 s.

nervous system (PNS) neurons [13]. However, clear evidence is not abundant for central nervous system (CNS) neurons. It would be reasonable to expect competition between Zn 21 and Cd 21 as both metals belong to the same IIB family in the periodic table of elements, indicating similar chemical properties. As depicted in Fig. 8, no significant increase was observed in [Ca 21 ] i (both cytoplasmic and nuclear concentrations) when Me5 neurons were pretreated with Zn 21 and then challenged by Cd 21 in the continuous presence of Zn 21 (n57). Zn 21 itself produced a delayed rise in [Ca 21 ] i on its own as has been reported in Xenopus oocytes [23]. However, the increase in [Ca 21 ] i observed was less than 50 nM (n56) (not illustrated).

4. Discussion

4.1. Does Cd 21 affect neurons directly or indirectly? The neurotoxicity of Cd 21 is supported by various clinical reports: chronic exposure to Cd 21 impaired intelligence and school achievement in children, and impaired cognition in workers [6,16,29]. However, a number of reports indicate that Cd 21 does not directly affect neurons but damages them indirectly via vascular impairment. The administration of Cd 21 initially affects the integrity and permeability of the vascular endothelium and necrotic changes in nerve cells are only secondary to this effect [11,12,25,28,40]. Contrary to this idea, some reports have pointed out that Cd 21 can directly induce pathological changes in nerve cells, e.g. in cultured rat sensory ganglion neurons [35]. And according to Kasuya et al. [22], Cd 21 not only suppressed the outgrowth of cultured cerebellar neurons but also caused degenerative changes to occur in these neurons. In addition, it has been proposed that it is a combined action of Cd 21 on blood vessels (ischemia) and direct Cd 21 toxicity on neurons which is responsible for the toxicity of Cd 21 on the nervous system [3]. The present work was carried out to re-evaluate the direct effect of acute Cd 21 application on CNS neurons using dissociated mesencephalic trigeminal (Me5) neurons

Fig. 8. Action of Zn 21 on the intracellular Ca 21 level of a Me5 neuron. Zn 21 antagonizes Cd 21 and inhibits elevation in [Ca 21 ] i . Extracellular applications of Zn 21 and Cd 21 are indicated by horizontal bars.

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which were devoid of the vascular component and suitable for the purpose. The results indicate that there is toxicity of Cd 21 due to a direct action on nerve cells.

4.2. Cd 21 -induced necrosis It was shown that morphological changes induced by 21 Cd in Me5 neurons (Fig. 1) were mediated by a sustained increase in [Ca 21 ] i (Figs. 3 and 4), because such changes were prevented when the elevation in [Ca 21 ] i was inhibited by the Ca 21 chelator BAPTA (Fig. 6). The morphological changes occurred predominantly to the cytoplasm: cytoplasmic granulation and vacuolization and cell swelling (Fig. 1). Also, some changes occurred to the nucleus, i.e. pyknosis. Finally, cell lysis or explosion occurred in Me5 neurons in extreme cases. These degenerative features of Me5 neurons are in agreement with the signs of necrosis reported in trigeminal and dorsal root ganglion neurons exposed to Cd 21 [3,13]. In addition, the results obtained by the TUNEL technique, which specifically detects apoptotic nuclear morphology, support necrosis rather than apoptosis as the process occurring in Me5 neurons (Fig. 2).

4.3. Possible mobilization mechanism of intracellular Ca 21 by Cd 21 In cat adrenal chromaffin cells, micromolar concentrations of Cd 21 were effective in depolarizing membrane potentials either by increasing the Na 1 influx [42] or by decreasing the K 1 conductance [31]. The membrane depolarization may increase the Ca 21 influx through voltage-gated Ca 21 channels. On the other hand, the same group obtained the data that 1 mM Cd 21 , a sufficient concentration for blocking voltage-dependent Ca 21 channels, caused a slow increase in [Ca 21 ] i [42]. They suggested that Cd 21 , at very high concentrations (1 mM), directly crossed the membrane and induced intracellular Ca 21 mobilization from the IP3 -sensitive Ca 21 stores via production of inositol 1,4,5-trisphosphate (IP3 ) [4,5,23,30,43]. It is to be noted that the Cd 21 influx at 1 mM was inhibited in cat adrenal chromaffin cells by Zn 21 as shown in the present study (Fig. 8), while depolarization-induced increase in [Ca 21 ] i was only partially suppressed by Zn 21 [42]. The protective effect of Zn 21 against Cd 21 has been reported for toxicity [2,9,13,15] and even for carcinogenicity [1,39].

4.4. A rapid examination system for checking neurotoxicity It takes time for conventional animal experiments to test the neurotoxicity of metals, as they can not easily invade the central nervous system (CNS) because of the blood– brain–cerebrospinal fluid barrier [34]. Indeed, the toxicity

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of Cd 21 on the nervous system is not easy to evaluate in human and animal studies. For instance, when rats were injected with Cd 21 and examined after a short period (up to 6 days), the metal did not enter the brain parenchyma [34]. However, in the cases of chronic administration of Cd 21 (e.g. for longer than 2 months), a considerable Cd 21 accumulation has been observed in the brain [8,21,33]. The present method, which directly applies Cd 21 to dissociated neurons, offers a rapid examination system for checking neurotoxicity of possible adverse substances.

Acknowledgements The author is grateful to The Central Research Laboratories at Fukui Medical School for the confocal laser scanning microscope system and Mrs. Junko Yamamoto for her technical assistance. Dr. A.J. Pennington at Department of Neuroscience, Edinburgh University Medical School and Dr. S. Macmillan at Quintiles Scotland Limited are also appreciated for revising the manuscript.

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