Yasuo Suzuki

Wilson's disease gene is homologous to hts causing abnormal copper transport in Long-Evans cinnamon rats.

BACKGROUND/AIMSnThe Long-Evans Cinnamon (LEC) mutant rat shows an excess copper accumulation in the liver and low serum ceruloplasmin activity. The disorder is controlled by a single autosomal recessive gene designated as hts. Wilsons disease is an autosomal recessive disorder of copper metabolism characterized by abnormal copper accumulation in the liver and low serum ceruloplasmin activity. The gene responsible for Wilsons disease has recently been isolated. The present study was designed to examine whether the LEC rat is an ideal animal model for Wilsons disease from a genetic point of view.nnnMETHODSnFor chromosomal mapping of hts, genetic linkage analysis using rat microsatellite marker loci was performed. Furthermore, cosegregation between hts and a rat counterpart of the Wilsons disease gene was analyzed.nnnRESULTSnhts was finely mapped to rat chromosome 16. Complete cosegregation between hts and a rat counterpart of the Wilsons disease gene was detected.nnnCONCLUSIONSnhts is likely to correspond to a rat homologue of the Wilsons disease gene. The present results allow us to propose that the LEC rat is an ideal animal model for Wilsons disease.

Hair Mercury Levels of Residents in China, Indonesia, and Japan

The authors used gold-amalgamation cold-vapor atomic absorption spectrometry and ECD-gas chromatography to analyze total mercury and methylmercury levels in hair samples obtained from 362 residents in Harbin, China; Medan, Indonesia; and Tokushima, Japan. In this study, the authors initially questioned whether mercury levels in hair differed among different study areas, and if there were differences, they questioned the contributing factors. In the three countries surveyed, total mercury and methylmercury levels in hair were lowest in residents of China and were highest in residents of Japan. In the district of Tokushima, Japan, total mercury and methylmercury levels were highest in the coastal district, followed by the middle district; the lowest levels occurred in the mountainous district. In Japan, an individuals total mercury level correlated very closely with that persons methylmercury level; in China and Indonesia, the correlation between these 2 parameters was low. No subjects in China or Indonesia had high levels of methylmercury in hair; this was true even if their total mercury levels were high. This finding suggests that the high total mercury levels observed in some residents of China and Indonesia reflected exposure to inorganic mercury. In Japan, mercury (especially methylmercury) levels in hair samples were quite high. Fish and shellfish, caught in seas uncontaminated by human activity, appeared to be major sources of the high levels of hair mercury in Japanese subjects.

Determination of 8-hydroxy-deoxyguanosine formation in rat organs: assessment of paraquat-evoked oxidative DNA damage.

Paraquat has previously been shown to cause the oxidative damage to DNA in variety of cells and tissues. However, although paraquat‐evoked strand breaks has been extensively studied to assess the DNA damage, the effect of paraquat on base modifications, another marker for the oxidative damage, has not yet been investigated. To further characterize paraquat‐evoked DNA damage, the effect of paraquat on 8‐hydroxy‐deoxyguanosine (8‐OH‐dG) formation in various rat organs was examined. Paraquat markedly increased 8‐OH‐dG contents in various organs, particularly in brain, lung and heart, and this increase reached the maximum levels at 5 days after the drug administration. In contrast, the formation of 8‐hydroxy‐guanosine (8‐OH‐G), a marker for the oxidative damage to RNA, was not significantly affected by paraquat. These results indicate that paraquat causes base modifications as well as strand breaks as a consequence of the oxidative damage to DNA.

Role of Copper Accumulation in Spontaneous Renal Carcinogenesis in Long-Evans Cinnamon Rats

Spontaneous renal cell tumors in totals of 223 male and female Long‐Evans Cinnamon (LEC) rats of 51–120 weeks old, 157 male F344 rats of 51–120 weeks old, and 14 male Long‐Evans Agouti (LEA) rats of 51–70 weeks old were examined histologically. The incidences of renal cell tumors increased with age in male and female LEC rats, but no tumors developed in F344 or LEA rats. Dilated atypical tubules of the kidneys were observed at high incidence in aged LEC rats. Copper staining of LEC rat kidneys showed a positive reaction in proximal tubules of the cortex and the outer stripe of the medulla. The renal copper concentration of LEC rats reached a peak in the period of necrotizing hepatitis with renal tubular necrosis, and was higher than that in F344 rats for up to 106 weeks. In contrast, the renal iron concentration of LEC rats was lower than that in F344 rats except in the period of necrotizing hepatitis. Long‐term treatment of LEC rats with d‐penicillamine, a copper‐chelating agent, inhibited accumulation of copper, but not iron, in the kidneys, and inhibited the development of karyomegaly of proximal tubules and dilated atypical tubules. These results suggest that persistent copper accumulation after toxic necrosis of tubules is the major cause of spontaneous renal carcinogenesis in LEC rats.

Adsorption and elution of metals on hair

The adsorption of zinc and lead on hair was dependent on the acidity of the hair and/or the medium in which the hair sample was immersed, suggesting that hair is an ion exchanger. The pKa was estimated to be between 4.5 and 5.0. The coexistence of mercuric ion or PCMB reduced zinc adsorption by only a few percent, whereas zinc inhibited mercuric ion adsorption to a greater extent. These facts suggest that the binding sites in hair for metals are located on functional groups like carboxyl groups rather than sulfhydryl groups.The removal and/or elution of metals from hair were observed for 18 elements by various washing procedures. By treating hair with a water solution of detergent, alkaline metals were eluted to a great extent, whereas alkaline earth metals were eluted to some extent. The other metals did not vary with any procedures tested.

Trace element contents in hair of residents from Harbin (China), Medan (Indonesia), and Tokushima (Japan).

The concentrations of 19 trace element in hair samples from 1273 residents of Harbin (China), Medan (Indonesia), and Tokushima (Japan) were measured by inductively coupled plasma emission spectrometry. The mean concentrations of Ba, Ca, and Se were significantly higher in the Harbin hair samples when compared to those from Medan, but Al, B, Cu, Fe, Mn, Na, Pb, Ti, Zn, and K were significantly higher in Medan than in Harbin hair samples. The differences in the mean concentrations of As, Cr, Mg, P, Sn, and Sr between the Medan and Harbin lots were not significant. In the Tokushima hair samples, Na and K were significantly higher, but As, B, Ba, Ca, Cr, Mg, Mn, Pb, Sn, Sr, and Se were significantly lower than in the Harbin hair samples. The differences in the mean concentrations of Al, Cu, Fe, P, Ti, and Zn between Harbin and Tokushima were not significant. In the Medan hair samples, Al, As, B, Ba, Ca, Cr, Cu, Fe, Mg, Mn, Pb, Sn, Sr, Ti, and Zn were significantly higher, but P and Se were significantly lower than in Tokushima hair samples. Differences in mean concentrations of Na and K between Tokushima and Medan were not significant.

Quantitation of chlordane residues in mothers' milk.

Chlordane residues were determined in human milk samples. Quantitative analysis was conducted for six residual compounds in the milk of 29 healthy Japanese donors who had experienced no occupational exposure to chlordane. All six compounds were detected in the milk from each donor. Geometric mean levels in whole milk (ng/ mL) and milk fat (ng/g), respectively, were:trans-nonachlor 0.55, 15.7; oxychlordane 0.39, 11.5;cis-nonachlor 0.14, 4.00;cis-chlordane 0.09, 3.08;trans-chlordane 0.04, 1.20; heptachlor epoxide 0.66, 20.0.In terms of the concentration (in both the whole milk and fat), a significant positive correlation was noted between any two compounds oftrans-nonachlor,cis-nonachlor, and oxychlordane. The residue level of each of these compounds had a significant positive correlation with the lipid content of the milk.The level of chlordane residues in human milk was not very high, but the data suggest widespread chlordane contamination among the general Japanese population.

Elevation of metallothionein gene expression associated with hepatic copper accumulation in Long-Evans Cinnamon mutant rat.

The mechanism of the metallothionein (MT) gene expression was investigated in a mutant rat, LEC, which exhibits an abnormal accumulation of copper in hepatocytes. The levels of MT mRNA were extremely high and correlated with the hepatic copper concentrations in LEC rat liver. Gel retardation assays in nuclear extracts from LEC rat liver showed an increase in the copper-dependent binding proteins, which bind to the metal responsive element (MRE) of the MT gene. These results suggest that the high intracellular copper accumulation results in the elevation of the MT gene expression through increasing a putative trans-activating factor in LEC rat.

Low susceptibility of Long-Evans Cinnamon rats to N-butyl-N-(4-hydroxybutyl)-nitrosamine-induced urinary bladder carcinogenesis and inhibitory effect of urinary copper

We studied the susceptibilities to N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine (BBN)‐induced urinary bladder carcinogenesis of male Long‐Evans Cinnamon (LEC), F344 and Long‐Evans Agouti (LEA) rats. Male rats (n=21) were given 0.1% BBN in their drinking water from week 6, 8 and 10 for one week, and killed in week 56. The incidences of transitional cell tumors (papillomas plus carcinomas) in BBN‐treated LEC and F344 rats were 12% and 76%, respectively (P < 0.001, experiment 1), and those in LEC and LEA rats were 11% and 95%, respectively (P < 0.001, experiment 2). When male LEC and F344 rats were given 0.1% BBN in their drinking water for 7 days, the intake of BBN and the urinary concentration of its active metabolite, N‐butyl‐N‐(3‐carboxypropyl)nitrosamine (BCPN), were higher in the LEC rats (P < 0.01). The urinary pHs of untreated LEC and F344 rats were similar between week 6 and 30. The urinary copper concentration was lower in LEC rats before jaundice than in F344 rats, but its concentrations in 28‐ and 50‐week‐old LEC rats were 1.7 and 2.3 times those in F344 rats. In a two‐stage carcinogenesis study using F344 rats, i.p. injections of cupric nitrilotriacetate increased urinary copper excretion, and inhibited BBN induced bladder carcinogenesis. In a two‐stage carcinogenesis study using LEC rats, oral administration of D‐penicillamine decreased urinary copper excretion, and increased BBN‐induced bladder cancer, although the difference was not significant. These data show that LEC rats are resistant to bladder carcinogenesis and suggest that urinary copper has a significant role in their resistance.

Effects of d‐Galactosamine Hydrochloride and Partial Hepatectomy on Spontaneous Hepatic Injury and Hepatocarcinogenesis in Long‐Evans Cinnamon Rats

To examine the effect of nongenotoxic chemicals on hepatocarcinogenesis in Long‐Evans Cinnamon (LEC) rats, we gave 6‐week‐old male and female LEC rats (n=18) weekly subcutaneous injections of d‐galactosamine hydrochloride (GalN, 300 mg/kg) in 0.9% NaCl or only 0.9% NaCl for 50 weeks, and killed them in week 62. GalN‐treated male rats unexpectedly showed no lethal necrotizing hepatitis. GalN treatment increased the incidence of cholangiofibrosis in males and its severity in females, but did not cause significant increases of hepatocellular tumors in either sex. GalN treatment increased the 5‐bromo‐2′‐deoxyuridine (BrdU)‐labeling index of hepatocytes and plasma hepatocyte growth factor, and accelerated megalocytic alterations without reduction of the hepatic copper concentration. Next, male and female LEC rats were subjected to two‐thirds partial hepatectomy (PH) or sham hepatectomy in week 8 (n=12) or in week 14 (n=9), and killed in week 62. PH in week 14 inhibited lethal hepatitis, but PH in week 8 was less effective. PH reduced the hepatic copper concentration to half that of controls. The present data suggest that induction of hepatocyte regeneration by repeated injections of GalN, or by PH just before the onset of jaundice has a significant effect in prevention of hepatic injury of LEC rats, but not enhancement of spontaneous hepatocarcinogenesis.