Masanori Ando

Functional characterization of two variant human GSTO 1-1s (Ala140Asp and Thr217Asn).

Glutathione-S-transferase class Omega (GSTO 1-1) belongs to a new subfamily of GSTs, which is identical with human monomethylarsonic acid (MMA(V)) reductase, the rate limiting enzyme for biotransformation of inorganic arsenic, environmental carcinogen. Recombinant GSTO 1-1 variants (Ala140Asp and Thr217Asn) were functionally characterized using representative substrates. No significant difference was observed in GST activity towards 1-chloro-2,4-dinitrobenzene, whereas thioltransferase activity was decreased to 75% (Ala140Asp) and 40% (Thr217Asn) of the wild-type GSTO 1-1. For MMA(V) reductase activity, the Ala140Asp variant exhibited similar kinetics to wild type, while the Thr217Asn variant had lower V(max) (56%) and K(m) (64%) values than the wild-type enzyme. The different activities of the enzyme variants may influence both the intracellular thiol status and arsenic biotransformation. This can help explain the variation between individuals in their susceptibility to oxidative stress and inorganic arsenic.

Relationship between the development of hepato-renal toxicity and cadmium accumulation in rats given minimum to large amounts of cadmium chloride in the long-term: preliminary study.

Abstract We wished to clarify the relationship between the sensitivity to induce hepato-renal toxicity and the level of cadmium (Cd) in the organs of rats exposed to minimum to large amounts of cadmium chloride (CdCl2). For this purpose, groups of female Sprague-Dawley (SD) rats, each consisting of 24 animals, were fed diet containing CdCl2 at concentrations of 0, 8, 40, 200, and 600u2009ppm for 2, 4, and 8 months from 5 weeks of age. All surviving rats given 600u2009ppm Cd were killed at 4␣months because of deterioration of their general condition. Animals of this group showed anemia and decreased hematopoiesis in the bone marrow, in addition to reduction of cancellous bone in their femurs. Hepatotoxicity was observed after 2 months in the groups treated with 200u2009ppm. By 4 months, the rats in the 600u2009ppm group had developed periportal liver cell necrosis. Renal toxicity characterized by degeneration of proximal tubular epithelia was apparent in the groups treated with 200u2009ppm from 2 months, becoming more prominent in the high-dose rats at 4 months. Hepatic accumulation of Cd increased linearly with the duration of treatment. In contrast, the concentration of Cd in the renal cortex of rats treated with 600u2009ppm reached a plateau level of ∼250u2009μg/g within the first 2 months. The renal concentration of Cd in the 200u2009ppm group when renal toxic lesions were first detected at 2 months ranged from 104 to 244u2009μg/g. No renal lesions were observed in the 40u2009ppm group after 8 months, despite the presence of 91–183u2009μg/g of Cd in the kidneys. The results thus suggest that renal toxicity would not be induced by treatment with minimum amounts of CdCl2 for periods longer than 8 months, although accumulation of Cd might gradually progress. A further 2-year feeding study of CdCl2 and Cd-polluted rice is now in progress.

Effects of cadmium administration on the endogenous metal balance in rats

The concentrations of cadmium and other metal ions in selected organs, urine, and blood of female rats were measured after exposure to cadmium chloride through their diet or by oral or intravenous administration. The hematological and urinary variations were followed for 4 wk.Body weight gain and the weights of livers and kidneys from all treated groups were not significantly different from the controls. No gross morphological changes were observed in any of the tissues studied at necropsy.The accumulation of cadmium occurred in the liver and kidney. The zinc levels in these organs were elevated relative to controls, in all treated groups regardless of dose and exposure route. Copper was elevated in the liver, kidney, bone, and blood of animals subject to intravenous administration of cadmium. Hepatic iron was decreased in the dietary and orally treated groups, but was not affected in the intravenous study group. The level of magnesium in kidney was increased for all exposure routes, but that of liver was increased only in the intravenously injected groups. The changes in the concentrations of sodium, potassium, calcium, and phosphorus did not follow a specific pattern and varied from organ to organ, depending on the exposure route.The discussion includes a relationship between tissue injury and the alteration of tissue essential element concentrations.

In vitro metabolism of simazine, atrazine and propazine by hepatic cytochrome P450 enzymes of rat, mouse and guinea pig, and oestrogenic activity of chlorotriazines and their main metabolites

1. The in vitro metabolism of chlorotriazines, simazine (SIZ), atrazine (ATZ) and propazine (PRZ) in liver microsomes from rat, mouse and guinea pig and the oestrogenic activity of chlorotriazines and their main metabolites have been studied. 2. The formation rates of products in chlorotriazine metabolism were determined by HPLC. The principal reactions catalysed by the cytochrome P450 (P450) system were N-monodealkylation and isopropylhydroxylation in all liver microsomes. As a result, 2-chloro-4-ethylamino-6-amino-1,3,5-triazine (M1) (SIZ-M1 for SIZ and ATZ-M1 for ATZ) and 2-chloro-4-amino-6-isopropylamino-1,3,5-triazine (M2) (ATZ-M2 for ATZ and PRZ-M2 for PRZ), and 2-chloro-4-ethylamino-6-(1-hydroxyisopropylamino)-1,3,5-triazine (M3) (ATZ-M3 for ATZ) and 2-chloro-4-isopropylamino-6-(1-hydroxyisopropylamino)-1,3,5-triazi ne (M4) (PRZ-M4 for PRZ) were detected as the metabolites. N-bidealkylation was not found in this system. 3. The formation rates of N-deethylated metabolites (SIZ-M1 and ATZ-M2) were generally higher in mouse than in rat and guinea pig. The formation rates of N-deisopropylated metabolites (ATZ-M1 and PRZ-M2) in guinea pig were the lowest among the three animal species. The formation rates of isopropylhydroxylated metabolites (ATZ-M3 and PRZ-M4) were remarkably low in mouse compared with rat and guinea pig. 4. The enzyme kinetics of chlorotriazine metabolism were examined by Eadie-Hofstee analyses. Some species differences in Michaelis-Menten parameters for each metabolite were observed, and the ranking orders were varied among the metabolites. 5. The binding affinity of chlorotriazines (SIZ, ATZ and PRZ) and their metabolites (M1-4) for recombinant human oestrogen receptor-alpha was assayed using the fluorescence polarization method. The binding affinity of M2 was significantly higher than those of parent compounds and other metabolites, although the oestrogenic activity was remarkably low compared with that of 17beta-oestradiol (E2). 6. These results suggest that the pattern of metabolism of SIZ, ATZ and PRZ by the P450 system differs extensively among rat, mouse and guinea pig, and that M2 may be an activated metabolite of chlorotriazines.

Changes in rat liver cytochrome P450 enzymes by atrazine and simazine treatment

1. We examined the effect of two chloro-s-triazines (atrazine and simazine) on hepatic microsomal cytochrome P450 enzymes in rat. Rats were treated intraperitoneally with atrazine or simazine daily for 3 days with 100, 200 and 400 mumol/kg. 2. Among the P450-dependent monooxygenase activities, testosterone 2 alpha-hydroxylase (T2AH) activity in rat, which is associated with CYP2C11, was significantly decreased at all doses of atrazine and simazine. The levels relative to control activities were 59-46 and 60-32% respectively. Similarly, oestradiol 2-hydroxylase (ED2H) activity was also significantly decreased by 28-51% by atrazine and simazine at all doses. However, no change in CYP2C11 protein level by either chloro-s-triazine was observed. K(m) for T2AH was significantly increased only by simazine (200 mumol/kg), whereas the Vmax and Cl(int) for T2AH were significantly decreased by atrazine and simazine at all doses. 3. 7-Ethoxyresorufin O-deethylase (EROD), 7-methoxyresorufin O-demethylase (MROD) and 7-pentoxyresorufin O-depentylase (PROD) activities were significantly increased by 1.4-1.6-, 1.7-3.2- and 1.5-2.2-fold respectively, by both chloro-s-triazines at 200 or 400 mumol/kg. Lauric acid omega-hydroxylase (LAOH) was also increased by 1.4-fold by simazine at 200 and 400 mumol/kg. Immunoblotting showed that only simazine induces CYP1A2 and CYP4A1/2 protein expression. 4. The activities of 7-ethoxycoumarin O-deethylase (ECOD), bufuralol 1-hydroxylase (BF1H), chlorzoxazone 6-hydroxylase (CZ6H), testosterone 6 beta-hydroxylase (T6BH) and testosterone 7 alpha-hydroxylase (T7AH) were not affected by either chloro-s-triazine. 5. These results suggest that the pattern of changes in P450 isoforms by chloro-s-triazines differs between atrazine and simazine, that these herbicides change the constitutive and/or male specific P450 isoform(s) in rat liver, and that these changes closely relate to the toxicity of chloro-s-triazines.

Studies on the disposition of calcium in bones of rats after continuous oral administration of cadmium.

Abstract In order to study the fate of calcium in bones of rats after continuous oral administration of cadmium, the whole body retention, the excretion, and the distribution of 45Ca were investigated in rats treated with Cd. The biological half-lives of 45Ca were found to be remarkably shortened in Cd-exposed rats compared with normal control rats. The degrees of fecal and urinary excretion of 45Ca of Cd-exposed rats were higher than those of control rats. The radioactivity of 45Ca in the blood increased more markedly in Cd-exposed rats than in control rats, whereas the amount of 45Ca in bones of Cd-exposed rats decreased with the duration of exposure.

Irgasan DP 300 (5-chloro-2-(2,4-dichlorophenoxy)-phenol) induces cytochrome P450s and inhibits haem biosynthesis in rat hepatocytes cultured on Matrigel.

1. The effect of Irgasan DP 300 (5-chloro-2-(2,4-dichlorophenoxy)phenol) on cytochrome P450 (P450) induction and haem biosynthesis was studied in rat hepatocytes cultured on Matrigel. 2. Irgasan DP 300 significantly induced 7-benzyloxyresorufin O-debenzylase activity, followed by 7-pentoxyresorufin O-depentylase and 7-ethoxyresorufin O-deethylase activities. 4-Nitrophenol hydroxylase, testosterone 6 beta-hydroxylase and methoxyresorufin O-demethylase activities were also slightly increased. The maximum induction of these enzyme activities was obtained at the same concentration of 125 microM in the culture medium. 3. Immunochemical blots using anti-rat cytochrome P450 antibodies revealed that Irgasan DP 300 preferably induced CYP2B1/2 along with a slight increase in 3A. These results indicate that Irgasan DP 300 is a phenobarbital-type inducer. 4. In the absence of exogenous 5-aminolevulinic acid (ALA), slight increases in protoporphyrin IX (2.6-fold) and coproporphyrin III (1.3-fold) were observed in the Irgasan DP 300-treated cultures. In contrast, when 75 microM ALA was present, Irgasan DP 300 (250 microM) caused an extensive accumulation of uroporphyrin I (13-fold). 5. Irgasan DP 300 inhibited rat hepatic uroporphyrinogen III synthase in vitro. 6. These results indicate that Irgasan DP 300 produced accumulation of hydroxymethylbilane in rat hepatocytes by inhibiting uroporphyrinogen III synthase, and consequently an accumulation of uroporphyrin I.

Functional characterization of three human cytochrome P450 2E1 variants with amino acid substitutions

1. Cytochrome P450 (P450) 2E1 is a hepatic enzyme of importance for the metabolism of xenobiotics such as drugs and environmental toxicants. Genetic polymorphisms of CYP2E1 in 5-flanking and coding regions have been found previously in Caucasian and Chinese populations. 2. In order to investigate the effects of amino acid substitutions on the function of CYP2E1, the enzymes of all known CYP2E1 variants in the coding region (CYP2E1.2, CYP2E1.3 and CYP2E1.4) with Arg76His, Val389Ile and Val179Ile substitutions, respectively, as well as the wild-type CYP2E1 (CYP2E1.1) were expressed in COS-1 cells, and their chlorzoxazone 6-hydroxylation and 4-nitrophenol 2-hydroxylation activities were determined. 3. The protein level of CYP2E1.2 was reduced to 29% compared with that of CYP2E1.1. The profiles of the level of activity relative to CYP2E1.1 for chlorzoxazone 6-hydroxylation (300 µ M substrate) and 4-nitrophenol 2-hydroxylation (150 µ M substrate) were very similar. 4. Although the K m values were not significantly different among wild-type and variant CYP2E1s in any oxidation metabolism, the V max and V max / K m of CYP2E1.2 on the basis of the CYP2E1 protein level were 2.7-3.0-fold higher than those of CYP2E1.1. In contrast, the levels of CYP2E1 protein and catalytic activity of CYP2E1.3 and CYP2E1.4 were not affected by the corresponding amino acid substitutions. 5. The findings suggest that Arg76 is closely associated with the function of CYP2E1, and that the genetic polymorphism of CYP2E1 is one cause of interindividual differences in the toxicity of xenobiotics.

Studies on excretion and uptake of calcium by rats after continuous oral administration of cadmium.

Abstract The excretion and the uptake of Ca in rats were investigated in detail after the oral administration of CdCl 2 for various periods of time. The rats which had been pretreated with CdCl 2 for 1 month excreted 55% of orally administered 47 Ca in feces, whereas the rats which had not been pretreated with CdCl 2 for the same period excreted only 20% of the orally administered 47 Ca into feces. These facts indicate that the absorption of Ca from the gastrointestinal tract was lowered by the exposure of rats to Cd. Most of the excreted 47 Ca was found in feces, even when Ca was administered iv. Thus, the rats which had been pretreated with CdCl 2 for 3 months excreted 80% of the iv administered Ca into feces. Furthermore, the uptake of iv administered 47 Ca into bones was found to decrease up to 30% by the pretreatment of rats with CdCl 2 for 3 months. These observations support the hypothesis that Cd is one of the important factors in the development of the so-called “itai-itai” disease, which is a form of osteomalacia and an endemic disease in Cd-polluted areas in Japan.

The inhibition of vitamin D-stimulated intestinal calcium transport in rats after continuous oral administration of cadmium

Abstract In order to obtain further information on the inhibitory effect of cadmium on intestinal calcium transport, it was studied in rats after continuous po administration of cadmium. Calcium transport in the intestine of rats fed normal calcium diet was no different in control and cadmium-dosed rats which were not stimulated with 1α-hydroxy cholecalciferol (1α-OH-D 3 ). However, the intestinal calcium transport in rats stimulated with 1α-OH-D 3 remarkably increased in controls but not in rats exposed to cadmium. Intestinal calcium-binding activity in rats exposed to cadmium was much less than that of control animals. Intestinal calcium-dependent adenosine triphosphatase, alkaline phosphatase, and calcium absorption of control rats showed time-related changes after injection of 1α-OH-D 3 , but no changes were observed in intestine of rats exposed to cadmium. From these results, it is considered that cadmium inhibited the effects of 1α-OH-D 3 on the calcium absorption in the rats intestine.