Shinya Wakusawa

Long term effects of phlebotomy on biochemical and histological parameters of chronic hepatitis C

OBJECTIVE:There is considerable evidence that iron is a risk factor for liver injury in chronic hepatitis C. Known as iron reduction therapy, phlebotomy reduces serum ALT activity. This effect might continue with maintenance phlebotomy and result in slower progression of liver fibrosis.METHODS:We examined the biochemical parameters and liver histology of patients with chronic hepatitis C treated by maintenance phlebotomy. For biochemical evaluation, 25 patients were treated by initial phlebotomy to reduce serum ferritin levels to 10 ng/ml or less and then observed for 5 yr with maintenance phlebotomy to maintain the iron-deficient state. For histological evaluation, liver biopsies were performed before and after the study period in 13 of the patients. Thirteen patients who were virological nonresponders to interferon alone and had undergone second liver biopsies after more than 3 yr served as histological controls.RESULTS:Serum aminotransferase levels were decreased significantly by initial phlebotomy and remained at the same levels during the study period (p < 0.05). The grading scores were improved significantly in the study group (p < 0.05) and unchanged in the controls. The staging scores remained unchanged in the study group but were increased in the controls (p < 0.005). Disease progression was significantly different between the two groups (p < 0.05).CONCLUSIONS:These results suggest that phlebotomy with maintenance lowers serum aminotransferase levels, improves liver inflammation, and suppresses the progression of liver fibrosis in chronic hepatitis C.

Iron accumulation in the liver of male patients with Wilson's disease

OBJECTIVES:There is accumulating evidence that ceruloplasmin, a copper protein with ferroxidase activity, plays an important role in iron metabolism. The genetic disorder, aceruloplasminemia, can lead to tissue storage of iron as in hemochromatosis. Because most patients with Wilsons disease, a genetic copper toxicosis, have hypoceruloplasminemia, some could be affected by iron overload.METHODS:Four male patients with Wilsons disease were enrolled in this study of pre- and post-treatment iron metabolism.RESULTS:Pretreatment copper contents of the liver were high in all four male patients studied as diagnostic of Wilsons disease. Genetic analysis supported their clinical diagnosis of Wilsons disease without a background of hemochromatosis. Pretreatment serum ceruloplasmin levels were <20 mg/dl in all four patients. A standard penicillamine treatment for 3–8.5 yr further decreased their serum ceruloplasmin levels. Post-treatment serum ferroxidase activity was low as was the serum ceruloplasmin protein. Copper contents in the liver decreased after treatment in all subjects. In contrast, nonheme iron in the liver increased during treatment. Pretreatment liver specimens were positive for histochemical iron in two patients, and post-treatment specimens were positive in all four patients. In two patients, serum aminotransferase levels rebounded with elevation of serum ferritin concentration during the treatment period. Subsequent iron reduction by phlebotomy ameliorated their biochemical liver damage.CONCLUSION:Iron overload related to hypoceruloplasminemia may be clinically important, particularly in male patients with Wilsons disease.

A significant reduction in serum alanine aminotransferase levels after 3-month iron reduction therapy for chronic hepatitis C: a multicenter, prospective, randomized, controlled trial in Japan

BackgroundIncreasing evidence indicates that iron cytotoxicity plays an important role in the pathogenesis of chronic hepatitis C (CHC). However, the biochemical effects of iron reduction therapy on CHC remain to be confirmed in a controlled study. This study aimed to test whether iron removal by repeated phlebotomy improves serum alanine aminotransferase (ALT) levels in patients with CHC.MethodsPatients were randomly assigned to an iron reduction therapy or control group. The patients in the treatment group received 3-month iron reduction therapy by biweekly phlebotomy, while the patients in the control group were followed up for 3 months with regular blood tests alone.ResultsThirty-three patients completed the 3-month treatment, while 29 patients received the complete follow-up. The serum ALT levels were reduced from 118 ± 79 to 73 ± 39 IU/L in the treatment group, but did not change in the control group (106 ± 45 versus 107 ± 48 IU/L). Posttreatment enzyme activity was decreased significantly from the baseline. Furthermore, it was significantly lower than the 3-month control level. Although 5 patients withdrew from the study, none was affected by any side effects of repeated phlebotomy that required them to discontinue the treatment.ConclusionsThis short-term controlled trial demonstrated the biochemical efficacy and safety of iron reduction therapy for patients with CHC.

Increase in P-glycoprotein accompanied by activation of protein kinase Cα and NF-κB p65 in the livers of rats with streptozotocin-induced diabetes

It is known that protein kinase C (PKC) signal transduction is enhanced in a diabetic state, and that PKC activator phorbol esters increase the gene expression of MDR1 in human tumor cells. To clarify the expression of the liver transporters under diabetic conditions and the roles of PKCalpha and the transcription factor NF-kappaB, we investigated the expression levels of Mdr1a, Mdr1b, Mdr2, Mrp2, Bcrp, Bsep, Oct1, Oat2, and Oat3 transporters, PKCalpha, IkappaB, and NF-kappaB in the liver of rats with STZ-induced hyperglycemia. A selective increase in the gene expression of Mdr1b was detected by RT-PCR. Western blotting with C219 antibody revealed an increase in P-glycoprotein. Although the mRNA level of PKCalpha was not affected, translocation of PKCalpha to the microsomal fraction was detected. NF-kappaB p65, IkappaBalpha and IkappaBbeta mRNA levels were increased as was the level of nuclear NF-kappaB p65. From these findings, it was suggested that STZ-induced hyperglycemia caused the upregulation of Mdr1b P-gp expression through the activation of PKCalpha and NF-kappaB.

Compound overload of copper and iron in patients with Wilson's disease.

This review of the copper–iron interaction in Wilsons disease was mainly based on ten patients (three females and seven males) studied in our institutes because the genetic tests of ATP7B for Wilsons disease of primary copper toxicosis and HFE for hemochromatosis, the biochemical parameters of copper and iron, and morphological studies on biopsied liver specimens were complete. All patients had hypoceruloplasminemia and hepatic lesions compatible with Wilsons disease. One patient was homozygous and nine patients were compound heterozygous for the mutations in ATP7B, and all patients were free from the major mutation, C282Y, of HFE. The biochemical parameters of iron metabolism were not specific, except for serum ferritin concentration. Judging from the traditional criteria, seven patients had hyperferritinemia. Histochemical iron was stained in the livers of seven patients and histochemical copper was found in nine patients. Microanalysis was more sensitive than histochemistry, detecting copper and iron accumulation in the hepatocellular lipofuscin particles of all patients. Using an improved fixative, intralipofuscin distribution was found to be different between cuprothionein and iron complexes. Iron overload in Wilsons disease might be worsened after treatment because of the close relation to hypoceruloplasminemia, in which the iron efflux from the liver to the circulation is disturbed.

Ursodeoxycholic acid induces glutathione synthesis through activation of PI3K/Akt pathway in HepG2 cells

Ursodeoxycholic acid (UDCA) is widely recognized as an effective compound in the treatment of chronic hepatitis and is known to modulate the redox state of the liver accompanied by an increase of GSH. In the present study, to access the antioxidative effect of UDCA and to clarify the molecular basis of the action on GSH level, we evaluated its effects in HepG2 cells exposed to excessive iron. UDCA inhibited both a decrease in the GSH level and an increase in the reactive oxygen species caused by excessive iron in the cells. UDCA increased the gene expression of the catalytic- and modifier-units of glutamine-cysteine ligase (GCL), which is a key enzyme in GSH synthesis. We further investigated the effect of UDCA on the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, and obtained results showing that UDCA-induced increase in the GSH level was prevented by LY294002, a PI3K inhibitor. In addition, Western blot analysis of Akt showed that, while the total Akt level remained unchanged, the phosphorylated Akt level was increased by UDCA, and this increase was also prevented by LY294002. Moreover, UDCA promoted the translocation of a transcription factor, nuclear factor-E2-related factor-2 (Nrf2), into the nucleus, and this action was inhibited by LY294002. From these results, it was indicated that UDCA increased the GSH synthesis through an activation of the PI3K/Akt/Nrf2 pathway. This may be a primary mechanism of antioxidative action of UDCA concerned with its therapeutic effectiveness in chronic hepatitis.

Measurement of serum hepcidin-25 levels as a potential test for diagnosing hemochromatosis and related disorders

BackgroundIron overload syndromes include a wide spectrum of genetic and acquired conditions. Recent studies suggest suppressed hepcidin synthesis in the liver to be the molecular basis of hemochromatosis. However, a liver with acquired iron overload synthesizes an adequate amount of hepcidin. Thus, hepcidin could function as a biochemical marker for differential diagnosis of iron overload syndromes.MethodsWe measured serum iron parameters and hepcidin-25 levels followed by sequencing HFE, HJV, HAMP, TFR2, and SLC40A1 genes in 13 Japanese patients with iron overload syndromes. In addition, we performed direct measurement of serum hepcidin-25 levels using liquid chromatography–tandem mass spectrometry in 3 Japanese patients with aceruloplasminemia and 4 Italians with HFE hemochromatosis.ResultsOne patient with HJV hemochromatosis, 2 with TFR2 hemochromatosis, and 3 with ferroportin disease were found among the 13 Japanese patients. The remaining 7 Japanese patients showed no evidence for genetic basis of iron overload syndrome. As far as the serum hepcidin-25 was concerned, seven patients with hemochromatosis and 3 with aceruloplasminemia showed markedly decreased serum hepcidin-25 levels. In contrast, 3 patients with ferroportin disease and 7 with secondary iron overload syndromes showed serum hepcidin levels parallel to their hyperferritinemia. Patients with iron overload syndromes were divided into 2 phenotypes presenting as low and high hepcidinemia. These were then associated with their genotypes.ConclusionDetermining serum hepcidin-25 levels may aid differential diagnosis of iron overload syndromes prior to genetic analysis.

Biological Monitoring of Pyrethroid Exposure of Pest Control Workers in Japan

Biological Monitoring of Pyrethroid Exposure of Pest Control Workers in Japan: Dong Wang, et al. Department of Radiological and Medical Laboratory Sciences, Nagoya University Graduate School of Medicine—Synthetic pyrethroids such as cypermethrin, deltamethrin and permethrin, which are usually used in pest control operations, are metabolized to 3‐phenoxybenzoic acid (3‐PBA) and excreted in urine. Though 3‐PBA can be used to assess exposure to pyrethroids, there are few reports describing urinary 3‐PBA levels in Japan. This study aimed to investigate the seasonal variation of the exposure levels of pyrethroids and the concentration of urinary 3‐PBA among pest control operators (PCOs) in Japan. The study subjects were 78 and 66 PCOs who underwent a health examination in December 2004 and in August 2005, respectively. 3‐PBA was determined using gas chromatography‐mass spectrometry. The geometric mean concentration of urinary 3‐PBA in winter (3.9 µg/ g creatinine) was significantly lower than in summer (12.2 µg/g creatinine) (p<0.05). Geometric mean concentrations of urinary 3‐PBA in the spraying workers and the not‐spraying workers within 2 d before the survey were 5.4 µg/g creatinine and 0.9 µg/g creatinine for winter with a significant difference between the groups (p<0.05), and 12.3 µg/g creatinine and 8.7 µg/ g creatinine for summer (p>0.05), respectively. A significant association of 3‐PBA levels and pyrethroid spraying was thus observed only in winter. In conclusion, the results of the present study show that the exposure level of pyrethroids among PCOs in Japan assessed by monitoring urinary 3‐PBA was higher than that reported in the UK but comparable to that in Germany. Further research should be accumulated to establish an occupational reference value in Japan.

Mutational analysis of the MRP2 gene and long-term follow-up of Dubin-Johnson syndrome in Japan.

BackgroundRecent studies have indicated that dysfunction or loss of the multidrug resistance protein 2 (MRP2) is the molecular basis of Dubin-Johnson syndrome (DJS). To clarify the genetic basis of the disease and the long-term stability of serum bilirubin levels, we conducted a mutational analysis of the MRP2 gene and followed up serum bilirubin levels in Japanese DJS patients 30 years after they were originally diagnosed, based on traditional criteria.MethodsPatients were interviewed by telephone, and blood tests, including a genetic analysis of MRP2, were performed on the patients and family members who gave informed consent.ResultsOver the 30 years, hyperbilirubinemia remained unchanged in four of the five patients studied, while it worsened in 1 patient whose DJS was complicated by chronic hepatitis C. From an MRP2 gene mutational analysis, six mutations, including the novel mutation 1177C>T, were found. Three patients of a consanguineous family were homozygotes for three mutations (298C>T, 1967+2T>C, and 2439+2T>C). Two patients were compound heterozygotes (1177C>T/2302C>T and 1967+2T>C/2026G>C). A familial study showed no difference in serum bilirubin levels between mutant/wild heterozygotes and wild/wild homozygotes.ConclusionsThe hyperbilirubinemia of four Japanese patients with DJS, one of whom had a novel mutation, 1177C>T, of the MRP2 gene, had not worsened with aging.

Identification of a novel 2026G→C mutation of the MRP2 gene in a Japanese patient with Dubin-Johnson syndrome

AbstractDubin-Johnson syndrome is a recessive inherited disorder with conjugated hyperbilirubinemia caused by a dysfunction of multidrug resistance protein 2 (MRP2) on the canalicular membrane of hepatocytes. A mutational analysis of the MRP2 gene was carried out in three Japanese patients and their family members. In two patients, the homozygous mutations c.1901del67 and c,2272del168 were found. In the third patient, a −24C→T polymorphism and the two mutations c.1901del67 and 2026G→C were detected. The 2026G→C mutation was a novel mutation in exon 16 affecting the conversion of Gly676 to Arg676 (G676R) in the MRP2 protein, and was not detected in fifty healthy volunteers. The G676R mutation was located in the Walker A motif of the first nucleotide binding domain in the MRP2 protein, and it was suggested that the mutation induced the dysfunction of the MRP2 protein. It was concluded that the compound heterozygosity of the two mutations of the MRP2 gene in the third patient contributed to the induction of hyperbilirubinemia in this case.