ESR characterization and metallokinetic analysis of Cr(V) in the blood of rats given carcinogen chromate(VI) compounds
Introduction
It has been shown that both biological trace elements and reactive oxygen species are related to the developments of various diseases 1, 2. Most of them are in the paramagnetic states and thus electron spin resonance (ESR) spectrometry is used to measure them in chemical and biochemical specimens 3, 4. The disposition of paramagnetic substances in living animals and the relationship between such paramagnetic substances and several diseases have been studied insufficiently, because the available methods for measuring them dynamically is lacking. We have recently proposed an in vivo blood circulation monitoring-electron spin resonance (BCM-ESR) method, which enabled the real-time pharmacokinetic analysis of stable nitric oxide spin-labeled compounds in living rats [5]. To extend the usefulness of the BCM-ESR method, we have applied this method to examine the metallokinetics of paramagnetic metal ions.
Chromium (Cr) exists widely in nature at several oxidation states from Cr(II) to Cr(VI) [6]. Although Cr is established to be an essential bio-trace element in animals and humans with respect to glucose and lipid metabolisms [7], it has been found to be toxic and carcinogenic for humans in the case of high exposure 8, 9, 10, 11. In fact, epidemiological studies revealed that industrial workers exposed to Cr(VI) show higher incidence of respiratory cancer than the normal population 12, 13. In support of the observation, Cr(VI) compounds have been shown to be more toxic and carcinogenic than Cr(III) [14], since Cr(VI) readily enters the cells by sulfate anion transport system 15, 16. Thus Cr(VI) incorporated into the cells is reduced to Cr(V), Cr(IV), and stable Cr(III) by cellular reducing systems 17, 18, 19, 20. Among these Cr species inside the cells, Cr(V) has been shown to be a long-lived reactive intermediate. The formation of the paramagnetic Cr(V) has been identified by ESR during the reduction of Cr(VI) by biochemical reducing agents such as GSH 21, 22, 23, 24, 25, Cys 24, 26, 27, H2O2 [28], ascorbic acid 29, 30, 31, riboflavin [32] and NADPH-dependent flavoenzymes involving glutathione reductase 33, 34, lipoyl dehydrogenase 33, 34 and ferredoxin-NADP+ oxidoreductase 33, 34. In addition, the formation of Cr(V) was detectable in subcellular and cellular systems such as cytosol [35], microsomes 35, 36, 37, mitochondria [38], human red blood cells 39, 40, 41, rat thymocytes [42], and cultured Chinese hamster V-79 cells [43]. In the field of L-band ESR, Liu et al. (1994) detected first Cr(V) in whole living mice [44]. However, in the field of X-band ESR with higher detection sensitivity than L-band ESR, dynamic detection of Cr(V) in living animals has not been reported as far as we know.
We detected Cr(V) in the circulating blood of living rats given Cr(VI) compounds using the proposed in vivo BCM-ESR method [5]. The coordination structures of Cr(V) species in the blood were analyzed in terms of their characteristic g values in comparison with g values of several model Cr(V) complexes with different coordination modes and were indicated to have coordination structures around Cr(V) such as CrO(S2O2) and CrO(O4). Then, ESR signal decay curves were evaluated by the curve-fitting method using pharmacokinetic analysis which was named as `metallokinetic analysis'. This paper reports the bioinorganic chemistry of Cr in terms of the detection, the possible coordination structure of the Cr(V) species, and the metallokinetic analysis of Cr(V) species in the circulating blood of living rats following administration of Cr(VI) compounds.
Section snippets
Reagents
Pentobarbital (50 mg/ml) was purchased from Dainabot Co. (Osaka, Japan). Sodium dichromate, 2-mercaptoethanol and N-ethylmaleimido (NEM) were obtained from Nacalai Tesque, Inc. (Kyoto, Japan). Dithiothreitol and 2,3-dimercapto-1-propanesulfonic acid were from Boehringer Ingelheim Co. (Ingelheim, Germany) and Aldrich Chemical Co. (Milwaukee, WI, USA), respectively. Potassium dichromate, glutathione (reduced form), ascorbic acid and NADH (reduced form) were purchased from Wako Pure Chemical
BCM-ESR and metallokinetic analysis
When a rat was administered Cr(VI), and BCM-ESR was performed, the ESR signal due to Cr(V) was observed at both g=1.986 and g=1.979 in the circulating blood, as shown in Fig. 2, similarly to the signal patterns observed in the reaction of Cr(VI) and fresh blood or erythrocytes (Fig. 3(a), (b)). It is interesting to note that the observed spectral patterns and changes due to Cr(V) in the BCM-ESR were different for potassium and sodium salts (Fig. 2, Fig. 4) even though there was no difference in
Discussion
We found first the ESR spectrum due to Cr(V) in the circulating blood of rats given K2Cr2O7 or Na2Cr2O7 by i.v. injection, in which two distinct signals at g=1.986 and g=1.979 were observed (Fig. 2). The same ESR spectra were detected in the fresh blood of rats and erythrocytes treated with Cr(VI) compounds (Fig. 3(a), (b)).
The coordination modes corresponding to these two ESR signals were characterized by comparing the ESR g values of several model complexes with different coordination modes,
Acknowledgements
This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan to H.S.
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