Studies on Neurotoxicological Mechanism of Mercury Containing Mineral Medicines and Potassium Bromate

Autor:	Jiunn-Jye Chuu, 褚俊傑
Rok vydání:	2000
Druh dokumentu:	學位論文 ; thesis
Popis:	88 Although the clinical intoxication from the abuse of cinnabar (a natural occurring HgS) and Ba Paul San have been reported, their neurotoxicological mechanisms are still not known. Thus, we attempted to comparatively study their neurotoxicological effects on the vestibular ocular reflex (VOR) system of guinea pigs. The commercial compounds were orally administered (1.0 g / kg/ bw) to guinea pigs once every day for consecutive 7 days. The results obtained showed that HgS at a dose of 1.0g/kg induced a reversible caloric hyperfunction (40% appearance) and irreversible hypofunction pattern (20% appearance) of caloric test. Similar to HgS, cinnabar-B (low lead) also induced similar caloric dysfunction, while cinnabar A (high lead) and PbCl2 profoundly produced caloric hyperfunction and hypofunction. Moreover, Ba Paul San-A and -B severely caused 50% of caloric hypofunction and 50% of no response. Monitoring the Hg contents of whole blood and cerebellum revealed that an increase in Hg contents was correlated with their neurotoxicological effects on VOR system, indicating that the inorganic and insoluble form of HgS or cinnabar could be absorbed from the gastrointestinal tract and detectable in the brain. Although morphological studies showed intact contour in the vestibular labyrinth, the enzymatic Na+/K+-ATPase activity of cerebellum was significantly inhibited by the HgS, cinnabar-A and Ba Paul San-A. Moreover, NADPH-diaphorase activity and cerebellar nitric oxide production also significantly increased. These findings suggest that the decreased Na+/K+-ATPase activity and the increased nitric oxide level in the cerebellum may be responsible for inducing dysfunctions of VOR system in guinea pigs treated with HgS and cinnabar. Neurotoxicity of mercury sulfide in the vestibular ocular reflex system of guinea-pigs A traditional Chinese mineral medicine Cinnabar, naturally occurring mercuric sulfide (HgS) is still occasionally prescribed, but the neurotoxic effects of HgS has not been elucidated. In this paper, an animal model of the purified HgS intoxication was established in guinea pigs in order to study neurotoxicity and pathophysiology of the vestibular ocular reflex system (VOR). Guinea-pigs were dosed with HgS by gastric gavage (0.01, 0.1 and 1.0 g /kg/day) for consecutive 7 days. By means of caloric testing coupled with the electronystagmo- graphic (ENG) recording in guinea-pigs, we have found that HgS at a dose of 0.1g/kg induced reversible caloric hypofunction pattern and at a higher dose of 1.0g/kg induced irreversible hypofunction of caloric test. Monitoring the mercury contents of various tissues (blood, kidney, liver and cerebellum) by continuous flow and cold vapor atomic absorption spectrometry (AAS) revealed that a certain amounts of HgS could be absorbed from the gastrointestinal tract and detectable in these tissues. In addition to the induced dysfunction of VOR system, HgS also caused disturbance of motor performance in guinea-pigs. In enzyme assay, Na+/K+-ATPase activity of cerebellum was also significantly inhibited by HgS. Morphological studies showed that partial cell lose only in the cerebellar Purkinje cells layer, but not in the granule cell layer, neither in the vestibular labyrinth. All of these findings suggest that cerebellar Purkinje cells are the sensitive target site responsible for HgS inducing dysfunctions of both VOR system and the motor performance in guinea-pigs. Thus, it is concluded that caloric test coupled with ENG recording in VOR system are certainly a sensitive biomarker for monitoring the neurotoxicity of HgS. Abnormal auditory brainstem responses in mice treated with mercurial compounds: Involvement of excessive nitric oxide In this paper, we attempted to establish a mouse model for monitoring oto-neurotoxicity of mercurial compounds. Mice were dosing with the sublethal doses of HgS (0.1 and 1.0g/ kg) or MeHg (0.2, 2.0 and 10mg/kg) by gastric gavage for consecutive 7 days. Analysis of the hearing threshold indicated that MeHg (2.0 or 10 mg/kg) can significantly elevated the threshold but MeHg (0.2mg/Kg) and HgS (0.1 or 1.0g/kg) can not. Recordings of auditory brainstem responses (ABR) showed that either MeHg- or HgS-treatment induced a significant prolongation of wave I-V and the interwave latencies. This ototoxicity of MeHg persisted even after 11 weeks discontinuous administration but that of HgS completely restored after 5 weeks discontinuous administration, which were intimately correlated with the disposition of Hg in the brain tissues. Moreover, the inhibition of Na+/K+-ATPase activity and nitric oxide overproduction in the brainstem are apparently related with the observed oto-neurotoxicity. Based on these findings, we conclude that the functional disturbance of brainstem induced by mercurial compounds may account, at least in part, for their oto-neurotoxicity. Neurotoxicity and tissue Hg contents in rats induced by mercuric sulfide and methyl mercury In this study, we compared the absorption, tissue Hg contents and neurotoxicity of two mercurial compounds, methyl mercury (MeHg) which is soluble and organic, and mercuric sulfide (HgS) which is insoluble and inorganic. Since HgS is naturally found in cinnabar, a substance used in Chinese mineral medicines, and relatively lacks of toxicology information, it is important for us to investigate its toxic effects. Both compounds were administered orally to male rats for 5 and 14 days with assays conducted at these times as well as 14 days after discontinuous administration. Neurotoxicity was assessed using five neurophysiological and neurobehavioral parameters. The results obtained showed that MeHg (2.0mg/kg) prominantly caused reduction (35% as compared to initial) of motor nerve conduction velocity (MNCV), prolongation (240% as compared to initial) of tail flick responses, incomplete restoration from the suppressive muscle action potentials, impairment of rota-rod motor performance (decrease 77%) effects and inhibition of Na+/K+-ATPase activity of sciatic nerve; The latter three toxicities were relatively irreversible. By contrast, the former three toxic effects of HgS were rather unremarkable and the latter two toxic effects were moderately and reversibly. The blood Hg levels were found to be correlated with the degree of toxicities of these two Hg compounds. These findings indicate that the insoluble HgS can still be absorbed from gastro-intestinal (G-I) tract and distributed in various tissues including the brain which is correlated with the observed toxities. The differential profile of neurotoxic potencies and reversibility of these two Hg compounds in this rat model provides us a better understanding on the toxicokinetics of HgS (an ingredient occasionally prescribed in Chinese mineral medicine). It is estimated that the toxic effect of HgS is ranging about one thousandth or less than that of MeHg. Prevention of pentobarbital tolerance by mercuric compounds in mice: Inhibition of Na+/K+-ATPase activity involved Prevention of Pentobarbital Tolerance by Mercuric compounds in Mice: Inhibition of Na+/K+-ATPase Activity Involved.The purpose of this study is to explore the possible mechanism of hypnosis-enhancing effect of HgS or cinnabar (a traditional Chinese medicine containing more than 95% HgS) in mice. Whether the component of Hg or sulfide is essential for their effects, the conventional methyl mercury (MeHg), NaHS and ZnS were used for comparative study. The main finding of this study is that a short period (7 days) of administration to mice with a nontoxic dose of mercurial compounds (HgS or cinnabar) and sulfide (NaHS or ZnS) not only significantly enhanced pentobarbital-induced hypnosis but also prevented tolerance induction after 7-day administration of pentobarbital. Furthermore, 10- and 35- days after the last administration of mercurial compounds and pentobarbital, the hypnotic response to pentobarbital restored normally in the control groups and the enhancing hypnotic effect of mercurial compounds still sustained. These effects of mercurial compounds correlated their accumulation in the brain and apparently related with the decrease in Na+/K+-ATPase and Ca2+-ATPase activities in the cerebral cortex. Further experiment indicated that mercuric compounds can decrease the blood nitric oxide which may be involved in the process of hypnosis-enhancing effect. Taken together, these findings indicated that the mercurial compounds including cinnabar, exert a long-lasting enhancing hypnotic activity and the nontoxic ZnS is perhaps useful for delaying or preventing pentobarbital-tolerance. Effects of methyl mercury, mercuric sulfide and cinnabar on active avoidance responses, Na+/K+-ATPase activities and tissue mercury contents in rats This study compared neurobehavioral toxicities of three mercurial compounds: methyl mercury (MeHg) is soluble and organic, while mercuric sulfide (HgS) and cinnabar (naturally occurring HgS) are insoluble and inorganic. Cinnabar, one of Chinese mineral medicine, is still used as a sedative in some Asian countries with relatively lacks of toxicological information. These mercurial compounds were administered intraperitoneally (MeHg, 2 mg/kg) or orally (HgS and cinnabar, 1.0 g/kg) to male rats once every day for consecutive 13 days with assays conducted during or after discontinuous administration for 1h, 2, 8 and 33 weeks. Neurotoxicity was assessed by the active avoidance response and locomotor activity. The results obtained showed that MeHg and cinnabar prominently and irreversibly caused decrease of body weight, prolongation of latency for escape from electric shock, decrease of the percentage for conditioned avoidance response (CAR) from electric shock, impairment of spontaneous locomotion and inhibition of Na+/K+-ATPase activity of cerebral cortex. By contrast, HgS reversibly inhibited spontaneous locomotion and Na+/K+-ATPase activity. It is noted that HgS significantly decreased the latencies of escape from electric shock during administration period which lasted for 33 weeks after discontinuous administration. Since pretreatment of arecoline (cholinergic receptor agonist) but not fipexide (a dopaminergic receptor agonist) could significantly shorten the prolonged latencies for escape caused by MeHg and cinnabar, suggesting that the deficit in the active avoidance response perhaps, at least in part, mediated by the dysfunction of the cholinergic rather than dopaminergic system. Determination of Hg levels of the whole blood and cerebral cortex revealed that the tissue mercury content is highly correlated with the degree of neurobehavioral toxicity of these Hg compounds. These findings implicate that the insoluble HgS and cinnabar can still be absorbed from G-I tract and distributed to the brain. The possibility that contamination of other minerals in the cinnabar accounts for the greater neurotoxic effects than HgS is under investigation. Changes in vestibulo-ocular reflex induced potassium bromate and thioglycolate in guinea pigs Potassium bromate (KBrO3) is known to be an oxidizing agent that is used not only as a food additive, mainly in the bread-making process, but also as a neutralizer in hair curling set. The home permanent cold wave hair kits consist of the first solution, thioglycolate which changes the disulfide (S-S) bonds of hair keratins to S-H bonds and makes hair flexible, followed by oxidizing the S-H bond to orginal S-S bond by the second solution 2% KBrO3, thus stabilizing the curling of the hair. Although it has been shown that bromate poisoning could cause severe and irreversible sensorineural hearing loss as well as renal failure, the action mechanism of vestibulo-ocular reflex influenced by KBrO3 remains to be studied. By caloric test coupled with the electronystagmographic (ENG) recording, the results showed that KBrO3 (50mg/Kg, SC) induced reversible caloric hyperfunction (40% appearance) but especially caused irreversible hypofunction pattern (20% appearance) of caloric test while combined with thioglycolate (15mg/Kg, SC). Therefore, it is worth while to elucidate the sites of the toxic effects of KBrO3 on VOR systems. Our data also indicated that that KBrO3 combined with thioglycolate prominantly and irreversibly caused decrease of retention time on equilibrium rod and impairment of spontaneous locomotion, while no difference on Na+/K+-ATPase and Ca2+-ATPase activities of cerebellum. However, KBrO3 alone inhibited spontaneous locomotion and equilibrium performance were only found until consecutive 2 weeks treatment. In order to understand the mechinary underlying these neurobehavioral and enztmatic activities changes, we also studied the change in nitric oxide (NO) level and bromide ion content, revealed no difference between KBrO3 alone and KBrO3 combined with thioglycolate in the NO and bromide level of blood, however, the latter can significantly caused cerebellar NO overproduction. Base on our findings, we demonstrated that whether KBrO3 alone or KBrO3 combined with thioglycolate induce oto-neurotoxicity including central VOR disorder. Meanwhile, These findings suggest that the increased nitric oxide level in the cerebellum, may be in partial responsible for inducing dysfunctions of VOR system in guinea pigs treated with KBrO3 combined with thioglycolate. The detrimental effects of potassium bromate and thioglycolate on auditory brainstem response of guinea pigs Although it has been shown that bromate poisoning could cause severe and irreversible sensorineural hearing loss as well as renal failure, the action mechanism of bromate-induced oto-neurotoxicity especially its combination with thioglycolate remains to be studied. In this study, we attempted to investigate the toxic effects of KBrO3 in combination with or without thioglycolate on the auditory brainstem response (ABR) system in the guinea-pigs which was claimed to be very susceptible to the xenobiotics. In a preliminary test, we have found that after consecutive 2 weeks administration, KBrO3 caused a significant prolongation of wave I-III and the interwave latencies of ABR as well as significantly elevated the threshold of hearing, suggesting that the conduction velocity of the peripheral auditory nerve was delayed. By contrast, the absolute latency of wave IV/V and the interwave latency of wave III-V were not significantly prolonged, suggesting that KBrO3 had no effect on the brainstem. This oto-neurotoxic effect of KBrO3 was markedly enhanced by combining with thioglycolate. Our data also indicated that KBrO3 combined with thioglycolate but not KBrO3 alone prominantly caused a decrease of body weight. However, enzymatic actitivies (including Na+/K+-ATPase and Ca2+-ATPase) and the level of nitric oxide (NO) have not seen significantly affected on the brainstem. Based on these findings, we tentatively conclude that whether KBrO3 alone or KBrO3 combined with thioglycolate induced oto-neurotoxicity majorly through the peripheral neurons system rather than via central brainstem intoxication.
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