* Department of Otolaryngology, National Taiwan University Hospital, Taipei, Taiwan; and
Institute of Toxicology and
Institute of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan
Received August 20, 2001; accepted January 23, 2002
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ABSTRACT |
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Key Words: caloric test; cerebellum; cinnabar; mercuric sulfide; Na+/K+-ATPase; vestibulo-ocular reflex.
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INTRODUCTION |
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The VOR system is an important contributor to stable vision during natural activity. It is a simple reflex arc, transmitting the velocity command from the vestibular nerve to the ocular motor neurons to ensure that eye velocity matches head velocity. Recent investigations of eye movements have revealed striking modifications to VOR gain (maximum slow-phase velocity/maximum head velocity) by vision. As a result, the eyes remain stationary with respect to the world, and images of the world are stable on the retina. The function of VOR system of the guinea pigs can be examined by the caloric test coupled with electronystagmographic (ENG) recordings (Young et al., 1991). Through the pattern of caloric nystagmus, lesions in the vestibular labyrinth, vestibular nuclei, and/or their connections to the cerebellum can be assessed (Vibert et al., 1993
). For example, lesions of the brainstem typically decrease the gain of VOR or spare it. However, increased VOR gain indicates a sign of cerebellar disease that has been attributed to a loss of inhibitory effects of the archi-cerebellum on the vestibular nuclei (Baloh et al., 1979
; Sharpe et al., 1981
). Thus, an animal model has been established to test the function of the VOR system and its higher inhibitory center at the cerebellum.
The histopathological study of the temporal bones and cerebellum was conducted and the colorimetric technique used to monitor the Na+/K+-ATPase activity of the cerebellum, following oral administration of mercuric compounds for 7 consecutive days. Additionally, nitric oxide analyzer (NOA) was employed to determine the nitric oxide content of the blood and cerebellum. Finally, mercury levels in whole blood and cerebellum were determined and correlated with their toxic effects on the VOR system. It is believed that this sensitive monitoring model system is useful not only to provide experimental information for the legislative regulation of these Chinese mineral drugs, but also for clinical assessment of the intoxication contents of cinnabar present in traditional Chinese medicines.
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MATERIALS AND METHODS |
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Caloric test.
For the vestibular function test, the guinea pig was restrained in a special holder, with its four legs free. A pair of clip electrodes was attached to the canthi of both temporal orbits, and a reference electrode was attached to the vertex at the midline (Young et al., 1991). A pathological nystagmus was considered if the presence of nystagmus was more than five beats (Wu and Young, 2000
). For the caloric test, 5 ml of ice water (0°C) was used to irrigate the ear canal for 5 s, with the animal in a normal prone position. Recordings were made with a two-channel ENG recorder (NY-13 Rion Co, Tokyo, Japan). The slow phase velocity (SPV) of caloric nystagmus was calculated from the conventional nystagmus curve. To analyze the caloric nystagmus curve, the calibration of eye movement in ENG recordings was set at 6°/10 mm (Stevens et al., 1995
). The SPV of each derived caloric nystagmus was calculated by dividing the amplitude of the slow phase by the duration and recorded as degrees/s. The mean SPV average in controlled guinea pigs was 6.8 ± 1.1°/s (mean ± SD). Those exceeding 9.0°/s (mean + 2 SD) were classified as hyperfunction, whereas those below 4.6°/s (mean - 2 SD) were hypofunction. Once there was no response in the caloric test, the animal was subjected to a second caloric test after an intermission of 5 min.
Histopathological studies.
To correlate the caloric responses of the animals treated by various mercurial compounds, three animals from each group were processed for histopathological study. Through deep anesthesia with sodium pentobarbital, the thoracic cavity was opened and the guinea pig was perfused through the left ventricle with saline solution, which was followed by 10% neutral buffered formalin intravital fixation. After complete fixation, the animal was decapitated and the cerebellum as well as temporal bones were harvested and placed in formalin solution. Following decalcification by trichloroacetic acid, neutralized by Na2SO4, dehydrated by a series of alcohol solutions, finally embedded in celloidin, and serially sectioned at 20 µm. Every 200 µm, two adjacent sections were stained with hematoxylin and eosin (Young et al., 1992). Multiple full coronal sections of the cerebellum were embedded in paraffin, sectioned at 6 µm, and stained with hematoxylin and eosin.
Assay of Na+/K+-ATPase activity.
Membrane Na+/K+-ATPase activities were assayed as reported previously (Liu et al., 1997; Rohn et al., 1993
). Briefly, the enzymatic activities were measured in triplicate in 96-covered-well microtiter plates at 37 ± 0.5°C on a shaker. Ninety µl of assay buffer (30% sucrose, pH 7.4) containing 2 µg of membrane protein was added to each well. The assay was started with the addition of 10 µl of ATP (final concentration 5 mM), producing a final reaction volume of 100 µl. The reaction was then terminated by the addition of 200 µl of malachite green plus ammonium molybdate (3:1). Pi (µM) released by substrate ATP hydrolysis was determined colorimetrically with a microplate ELISA reader (Dynatech MR 7000) at 630 nm. The specific ATPase activities were expressed as µM inorganic phosphate per milligram protein per 20 min. Values reported herein represent the mean and SE of at least three separate experiments.
Nitric oxide (NO) determination.
The cerebella were weighed and homogenized in the homogenate buffer (0.32 M sucrose-histidine buffer, pH = 7.4 ) and then centrifuged at 4°C for 20 min at 10,000 x g. For total protein denatured, 70% ethanol was added to the sample (the homogenate 1:1) overnight, then all samples were centrifuged at 4°C for 20 min at 12,000 x g. The supernatants of cerebellar cortex were collected and assayed for NOx by the NO/ozone chemiluminescence (NO-Analyzer 280A Sievers; McKee et al., 1994). Briefly, oxidation products (NO2- and NO3-) of NO were measured using a reaction vessel that contained a reducing system (0.1 M vanadium chloride, Aldrich Co., U.S.). Detection of NO was then completed by its reaction with ozone, which led to the emission of red light (NO + O3
NO2* +O2; NO2*
NO2 + hv). Standard curves were made prior to concentration (1, 5, 10, 15, and 20 µM NO), which were from freshly prepared solutions of NaNO2 (10 µl) in distilled water.
Determination of mercury accumulation.
Various tissues (500 µl of blood, 300 mg of cerebellum) were placed in a wide-mouth polyethylene scintillation vial, and 3 ml of a 3:1 mixture of concentrated nitric acid:70% perchloric acids was added along with 50 mg of vanadium pentoxide. The vials were capped and allowed to stand overnight at room temperature. Following predigestion, the capped vials were heated for 4 h on a shaking water bath at 68 ± 0.5°C, then uncapped and heated continually for 3 h. After cooling, five drops of 30% hydrogen peroxide were added, and the vials were again capped and allowed to stand overnight to complete the digestion. Suitable dilutions were made from the digested material, and the total mercury content was determined with a solution of 2% SnCl2 in 0.05 M H2SO4. The reduction with SnCl2 and total tissue mercury content analysis by cold-vapor atomic absorption spectrophotometry were performed with automated equipment (Hitachi, Model Z-8200 and HFS-2 Hydride formation system). The reducing agents and apparatus were the same as reported (Iverson et al., 1974) except that 1.0 ml of diluted digest was placed in the modified Erlenmeyer flask. Then 4.0 ml of the reduced mixture was added to the closed system through a flexible plastic tube sealed into the ball-joint cap and connected to a syringe type automatic pipette.
Statistical analysis.
Analysis of variance (ANOVA and 2) was used to evaluate the results. When ANOVA was significant, the significance between groups was assessed by means of ANOVA followed by Dunnett's t-tests for most testings. Only discrete, relevant caloric response variables were analyzed by
2 tests, with three degrees of freedom for the trend in binomial proportions after appropriate multivariate adjustments. It was considered to be significant when the p value was less than 0.05.
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RESULTS |
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Effects of mercuric compounds on caloric response.
Throughout the experiment, neither spontaneous nystagmus nor positional nystagmus was noted in guinea pigs of each group. Following administration for 7 consecutive days, HgS exerted a moderate deleterious effect on the caloric test (60% abnormality, including 40% hyperfunction and 20% hypofunction; Figs. 1B and 1C). However, the neurotoxic effects of cinnabar in six animals definitely provoked a higher percentage of abnormal caloric responses, including hyperfunction in four (67%) and hypofunction in two (33%; Figs. 1D and 1E
). Comparison of the abnormal caloric responses between the control group (0%) and the HgS group (60%) or cinnabar group (100%) revealed a significant difference (p < 0.05,
2 test; Table 1
). Eight animals treated with PbCl2 alone also disclosed 100% abnormality, including caloric hyperfunction in seven (88%) and caloric hypofunction in one (12%). Table 1
lists the corresponding duration and slow phase velocity of caloric nystagmus derived from guinea pigs in each group.
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DISCUSSION |
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The VOR system consists of a direct excitatory projection from the lateral semicircular canal to the medial vestibular nucleus, then to the ipsilateral medial rectus subnucleus through the ascending tract of Deiters (McCrea et al., 1987). Another direct excitatory pathway projects into the contralateral abducens nucleus. The latter contains both motor neurons to the lateral rectus muscle and interneurons that go via the medial longitudinal fasciculus to the medial rectus motor neurons. If lesions occur on the vestibular labyrinth or brainstem, absent or decreased VOR gain (caloric hypofunction) will ensue, whereas increased VOR gain (caloric hyperfunction) attributes to central disinhibition due to lesions in the cerebellum. In this study, guinea pigs treated with either cinnabar or HgS in a similar dose (1.0 g/kg) resulted in caloric hyperfunction or hypofunction, and possible mechanisms of these results were explored.
First, vestibulotoxicity by mercurial compound was considered. It has been reported that intoxication of mercuric chloride in the inner ears of guinea pigs may damage labyrinthine blood vessels by causing swelling of the endothelial cells, mitochondrial disintegration, and sometimes protrusion of endothelial cell cytoplasm herniating into the blood vessel lumen. Chronic mercuric chloride intoxication resulted in distorted endothelial cells with an increase in cytoplasmic density (Anniko and Sarkady, 1977). Second, mercury deposits typically occurred in the cerebelli of methylmercury-poisoned rats and appeared in parts of the vestibular nerves, cochlear nerves, spiral ganglion, and stria vascularis. However, no pathological changes were detected in the vestibule (Igarashi et al., 1992
). In this study, we found that guinea pigs treated with HgS or cinnabar (1.0 g/kg) did not show marked change in the vestibular labyrinth under light microscopy (Fig. 2
). Toxic effects of these mercurial compounds on other tissue merits exploration. Accordingly, the symptoms of patients with organic mercury-compound poisoning in Niigata, Japan manifested as dizziness, nystagmus, and abnormal caloric response, and was attributed to lesions in the brainstem and cerebellum (Mizukoshi et al., 1975
). Our results also showed the distinguished lesions in the cerebellum of guinea pigs treated with HgS and cinnabar.
It has been suggested that the cerebellar lesion, combined with less involvement of the vestibular nuclei, may result in caloric hyperfunction, whereas lesion in the vestibular nuclei associated with cerebellar involvement exhibits caloric hypofunction (Fernandez and Fredrickson, 1964; Young and Nomura, 1995
). Thus, the signaling pathway of caloric response depends on the degree of involvement of the vestibular labyrinth, vestibular nuclei, and cerebellum. The caloric hyperfunction induced by cinnabar or HgS on the VOR system may be initiated from the sensitive higher center at the cerebellum and then progressively descend along this signaling pathway, resulting in caloric hypofunction.
Determination of the cerebellar mercury contents after treatment with HgS and cinnabar has been correlated with the abnormal caloric responses. The mercury accumulation in the blood and cerebellum of guinea pigs of the cinnabar group (840 ± 56.4 ppb and 400 ± 43.2 ppb, respectively) was more than that of the HgS group (350 ± 28.9 ppb in blood and 230 ± 27.8 ppb in cerebellum; Fig. 6). This result is correlated with the functional abnormality in caloric response, more severe in the cinnabar group than in the HgS group. Similarly, the number of Purkinje cells of the cinnabar group also had greater loss than those of the HgS group. The reason for a more severe cerebellar dysfunction caused by cinnabar is not known. We suspect that the existence of other elements such as lead in the cinnabar may enhance the solubility, absorption, and detrimental effect of cinnabar on the VOR system. Actually, PbCl2 by itself is also toxic to the VOR system (Table 1
).
Among various tissues, the cerebellar Purkinje cells were the most obvious staining targets for mercury accumulation (Warfvinge et al., 1992). In this study, it was also demonstrated that distortion or obvious loss in the Purkinje cell layer occurred following HgS or cinnabar treatment. Moller-Madsen (1991) reported that after a 20-day (20 mg/day) treatment of methylmercuric chloride, mercury deposits in the cerebellar cortex were restricted to Purkinje cells and Golgi cells. Similarly, in the spinal cord, the majority of mercury was located in the motor neurons, which inhibited VOR relay cells mediated through flocullar Purkinje cells (Doya, 2000
; Ito et al., 1970
; Snyder, 1999
). In addition, nodulo-uvular Purkinje cells inhibit vestibular nuclear cells that are projecting into the cerebellar cortex (Precht et al., 1976
). Additionally, degeneration of Purkinje cells (Figs. 3B and 3C
) may cause central disinhibition of the VOR system (Figs. 1B and 1D
) or abolish VOR adaptation (Figs. 1C and 1E
), resulting in abnormal caloric response (Robinson et al., 1976
).
Membrane Na+/K+-ATPase activity is essential to maintaining the Na+- and K+-gradient across the neuronal membrane. Notably, the inhibition of this enzymic activity can produce membrane depolarization, leading to the suppression of neuronal transmission (Albrecht and Hilgier, 1994; Anner et al., 1992
; Balestrino et al., 1999
). This study indicated that Na+/K+-ATPase activity of the cerebellum in the cinnabar group decreased more than that in the HgS group. In previous reports, the cerebellum was proved to be one of the most vulnerable regions, as it contains the highest deposition of mercury (Moller-Madsen, 1991
; Warfvinge et al., 1992
). Thus, it is proposed that HgS and cinnabar inhibit the cerebellar Na+/K+-ATPase, which may be responsible for inducing dysfunction of the VOR system of guinea pigs.
Nitric oxide (NO) was found to act as an essential factor in cerebellar functional modulation (Avrova et al., 1999). The regulation of Na+/K+-ATPase activity by NO and cyclic guanine monophosphate (cGMP) in various tissues including brain has been proposed (Lin et al., 1999
). However, its detailed role in the VOR system remains unknown. Figures 4 and 5
demonstrate that HgS and cinnabar reduced Na+/K+-ATPase activity and increased cerebellar NO production in guinea pigs. Although numerous action mechanisms have been suggested for methylmercury compounds including effects on neurotransmitter release, block of voltage-gated cationic channels, increase in intracellular calcium, and mitochondrial function (Atchison and Hare, 1994
), these effects probably induced the cascade of neurotoxic effect of mercury. Hence, the decreased Na+/K+-ATPase activity and the increased nitric oxide level in the cerebellum are suspected to induce dysfunction of the VOR system in guinea pigs treated with HgS or cinnabar.
In conclusion, we have demonstrated for the first report that the insoluble form of HgS and cinnabar can be absorbed from the gastrointestinal tract and distributed into the blood and cerebellum. The loss of Purkinje cells as well as the mercuric contents in the cerebellum correlates with the severity of abnormal caloric responses. The decreased Na+/K+-ATPase activity and the increased NO production in cerebellar tissue are proposed, at least in part, to be responsible for the dysfunction of the VOR system caused by HgS and cinnabar. The implication of these findings is that cinnabar, a naturally occurring HgS, which is used as a sedative in Chinese medicines, should be recognized as a potentially harmful substance.
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ACKNOWLEDGMENTS |
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NOTES |
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