Kiyohiko Dohi

ADENINE NUCLEOTIDE METABOLISM DURING HEPATIC ISCHEMIA AND SUBSEQUENT BLOOD REFLOW PERIODS AND ITS RELATION TO ORGAN VIABILITY

In an experimental model system for liver transplantation, the ability of the rat liver to synthesize ATP and to maintain adequate energy charge and total adenine nucleotides during restoration of hepatic blood flow following warm ischemia was found to determine tissue viability and survival of the animal. A portafemoral shunt prepared to relieve portal congestion enhanced the rate and extent of ATP resynthesis by the reflow following hepatic ischemia and this was accompanied by an increase in the survival rate of the rat.

Reliable Indices For The Determination Of Viability Of Grafted Liver Immediately After Orthotopic Transplantation Bile Flow Rate And Cellular Adenosine Triphosphate Level

One of the major problems accompanying liver transplantation is how to evaluate the viability of the grafted tissue at an early stage. The ability to assess immediate graft function would provide results useful in the determination of prognosis. The present study was undertaken to determine whether bile flow rates after liver transplantation were correlated with adenosine triphosphate levels and the survival of rats given transplants. In fresh-liver-transplanted rats, the one-week survival rate was 87%. The cellular ATP levels in the grafts decreased sharply prior to portal-venous declamping, but returned to nearly 80% of the normal level 4 hr after grafting, as did the total adenine nucleotide level and energy charge. When the grafts were subjected to warm ischemia for 15-min or 30-min periods prior to harvesting of the donor liver, the one-week survival rates decreased to 50% and 0%, respectively. In these cases, the levels of cellular ATP and bile secretion remained low and were proportional to the survival of the transplanted animals even 4 hr after transplantation. The relationship between the bile flow rates and the cellular ATP levels under various conditions revealed a good correlation, showing a saturation curve. The bile flow rates as well as the cellular ATP levels were therefore related to the survival rates of the transplanted animals. Thus it was shown in this experimental transplantation model that the monitoring of bile production after liver grafting is a useful indicator for assessing the extent of ischemic damage to the liver and for prognosis of the animal.

Changes in the levels of endogenous coenzyme Q homologs, α-tocopherol, and glutathione in rat liver after hepatic ischemia and reperfusion, and the effect of pretreatment with coenzyme Q10

The present study was undertaken to determine whether hepatic ischemia and the subsequent reflow of blood had any effect on the levels of endogenous coenzyme Q homologs, alpha-tocopherol, and glutathione, and whether coenzyme Q10 (6 mg/kg of body weight) altered these levels. Ischemia of the rat liver for 90 min resulted in decreases of 19.1 and 19.6% of endogenous alpha-tocopherol and total glutathione (GSH + GSSG) without significant changes in the levels of endogenous total coenzyme Q homologs (oxidized and reduced). Restoration of the blood flow resulted in marked decreases in endogenous coenzyme Q homologs, alpha-tocopherol, and total glutathione in the control group. In coenzyme Q10-treated animals, however, there were no changes in the levels of endogenous total coenzyme Q9, alpha-tocopherol, or total glutathione as well as in the level of the enhanced total coenzyme Q10 during the reperfusion period. On the other hand, decreases in alpha-tocopherol and total glutathione during the period of ischemia remained unchanged. These results are compatible with the assumption that cellular damage caused by hepatic ischemia can be explained by free radical reaction processes during ischemia and especially, reperfusion and suggest that exogenous coenzyme Q10 functions as an antioxidant with endogenous coenzyme Q homologs, alpha-tocopherol, and glutathione in lipid peroxidation during reperfusion.

Radiation Induction of Germline Mutation at a Hypervariable Mouse Minisatellite Locus

Paternal 60Co gamma-irradiation was tested for the induction of germline mutation at the mouse hypervariable minisatellite locus, Ms6hm. Male C3H/HeN mice were exposed to 3 Gy 60Co gamma-ray and mated with C57BL/6N females. Matings were made at 1-7, 15-21 and 71-77 days post-treatment to test spermatozoa, spermatids and spermatogonia stages. Reciprocal crosses were also made with irradiated C57BL/6N males. Southern analysis was carried out on DNA from parents and F1 mice. The paternal mutation frequencies per gamete of the Ms6hm locus were 8.3, 13, 28 and 15% for the C3H/HeN control, exposed spermatozoa, spermatids and spermatogonia stages, respectively. The paternal mutation frequencies per gamete were 7.7% for the C57BL/6N control and 13% for the C57BL/6N exposed spermatozoa stage. The increase in the paternal germline mutation frequency was statistically significant for C3H/HeN spermatids irradiation (p < 0.005). The induced mutation frequencies were of the order of 10(-1), and was too high to be accounted for by the direct action of radiation on the locus. These results suggest the presence of a previously unexpected mechanism of radiation induction of germline mutation. In addition, we demonstrate that the hypervariable minisatellite locus can serve as a sensitive monitor for genetic damages to germline cells.

Chemoprevention of N-Nitroso-N-methylurea-induced Rat Mammary Carcinogenesis by Soy Foods or Biochanin A

We examined the effects of soybeans, a soy product (miso) and biochanin A, an isoflavone derivative, on N‐nitroso‐N‐methylurea (MNU)‐induced rat mammary carcinogenesis. Seven‐week‐old female CD/Crj rats received a single i.v. dose (40 mg/kg body weight) of MNU. After administration of MNU, rats were fed diet containing 0% (control), 2% or 10% soybeans, or 10% miso as a soy‐supplemented diet, or 10 or 50 mg/kg biochanin A. All rats were observed for 18 weeks after MNU administration. At 18 weeks, the multiplicity (mean tumors/rat) of palpable mammary tumors was significantly decreased in the 10% soybean (1.1) and 10% miso (1.2) diet groups compared to the control (2.2) (P<0.05, respectively). In the biochanin A‐supplemented diet groups, the incidence (percentage of rats with tumors) was significantly decreased in the 50 mg/kg (32%) diet group compared to the control (80%) (P<0.01), and the multiplicity was significantly decreased in both the 10 mg/kg (0.7) and 50 mg/kg (0.5) diet groups compared to the control (2.2) (P<0.01 and P<0.001, respectively). The proliferative cell nuclear antigen labeling index of mammary tumors was significantly decreased in both biochanin A‐supplemented diet groups compared to the control. The present results indicate that soybeans, miso, and biochanin A are useful for the prevention of mammary cancer.

Polymerase chain reaction—single-strand conformation polymorphism analysis of polymorphism in DPA1 and DPB1 genes: A simple, economical, and rapid method for histocompatibility testing

A new technical trial was carried out to detect polymorphism in HLA-DP genes, based on the diversity in electrophoretic mobility of single-stranded DNA (single-strand conformation polymorphism, SSCP). Genomic DNAs from 31 cell lines homozygous for 2 and 14 different DPA1 and DPB1 alleles, respectively, and from peripheral blood cells of a normal individual homozygous for another DPB1 allele were subjected to polymerase chain reaction (PCR) to amplify the polymorphic exon 2 of DPA1 or DPB1 genes. The PCR samples were denatured by heating in the presence of formamide to obtain single-stranded DNA, electrophoresed in a neutral polyacrylamide gel, and visualized by silver staining. Allelic differences were detected by the distinctive electrophoretic pattern of each single strand, depending on the sequence-specific conformation. Fifteen DPB1 alleles showed 11 distinct electrophoretic patterns, leaving four allelic combinations not distinguished. These four allelic combinations could be further distinguished by using another couple of primers in PCR, with which a part of the exon was amplified, and by subsequent SSCP analysis. The use of four pairs of primers in PCR allowed for discrimination of all the 15 DPB1 alleles tested. Two allelic differences in exon 2 of DPA1 gene could be clearly demonstrated. In addition, putative new alleles of DPA1 and DPB1 genes were detected by SSCP analyses. The PCR-SSCP analysis is simple and rapid, requires neither radioactive materials nor restriction enzymes, and is expected to be a useful tool for investigating the fine HLA-matching required for clinical transplantation of organs.

Dose—response of a Radiation Induction of a Germline Mutation at a Hypervariable Mouse Minisatellite Locus

Dose-response of an induction of a germline mutation was studied at a hypervariable mouse minisatellite locus, Ms6hm, which consists of tandem repeats of a sequence motif GGGCA. Male C3H/HeN mice were exposed to various doses of 60Co gamma-ray and mated with unirradiated C57BL/6N female mice. Matings were done at various time after irradiation to assess the effects of radiation on spermatozoa, spermatids and spermatogonia. DNA samples of F1 offsprings were analysed by Southern blotting for the repeat length mutation at the Ms6hm locus. The mutation frequency per gamete of the paternal allele was 9.1% for the unirradiated control group. The spermatids stage was most sensitive to radiation and a statistically significant dose-response was observed. The mutation frequency of the paternal allele in F1 mice increased to 22% for 1 Gy, 28% for 2 Gy, and 28% for 3 Gy. The spermatogonia stage was less sensitive to radiation, and the mutation frequencies of the paternal allele were 14% for 2 Gy, and 16% for 3 Gy. The spermatozoa stage germ cells were also less sensitive and the frequency of mutation of the paternal allele increased to 14% for 3 Gy. However, these increases were statistically not significant. Possible mechanisms of radiation induction of germline mutation at the hypervariable minisatellite locus will be discussed.

Genetic alterations in thyroid tumor progression: association with p53 gene mutations.

To identify the genetic events that must be involved in thyroid tumor progression, we initially investigated p53 gene alterations in 10 papillary adenocarcinomas, 4 follicular adenocarcinomas, and 8 undifferentiated carcinomas. Base substitutional mutations in exons 5 to 8 and loss of heterozygosity (LOH) of the p53 gene were not detected in papillary or follicular adenocarcinomas. However, 7 of 8 undifferentiated carcinomas were carrying base substitutional mutations, and LOH was detected in 3 of 5 informative cases. Furthermore, to verify that the p53 gene alterations are truly involved in tumor progression, DNA from individual foci of the four undifferentiated carcinomas coexisting with a differentiated focus and from one follicular adenocarcinoma with an undifferentiated focus was analyzed by direct sequencing and polymerase‐chain‐reaction‐restriction‐fragment‐length polymorphism (PCR‐RFLP). Base substitutional mutations in the p53 gene from exons 5 to 8 were identified exclusively in the undifferentiated foci, but not in the differentiated foci. LOH was observed in 3 of 4 informative undifferentiated foci. In one of these positive cases, LOH was observed in both papillary adenocarcinoma and undifferentiated carcinoma. However, a p53 gene mutation at codon 248 was detected in the undifferentiated carcinoma but not in the papillary adenocarcinoma. The results imply that LOH occurs first in papillary adenocarcinoma followed by a p53 mutation during the transition from papillary adenocarcinoma to undifferentiated carcinoma. Maintenance of LOH during tumor progression excludes the possibility that these different histological foci are derived from different origins and represents molecular evidence that undifferentiated carcinoma is very likely derived from preexisting papillary adenocarcinoma. Furthermore, these results strongly suggest that the mutated p53 gene plays a crucial role in de‐differentiation during the progression of thyroid tumors.

Mutations in the RAD54 recombination gene in primary cancers

Association of a recombinational repair protein RAD51 with tumor suppressors BRCA1 and BRCA2 suggests that defects in homologous recombination are responsible for tumor formation. Also recent findings that a protein associated with the MRE11/RAD50 repair complex is mutated in Nijmegen breakage syndrome characterized by increased cancer incidence and ionizing radiation sensitivity strongly support this idea. However, the direct roles of BRCA proteins and the protein responsible for NBS in recombinational repair are not clear though they are associated with the recombinational repair complexes. Since RAD51 forms a complex with other members of the RAD52 epistasis group and with BRCA proteins, it is reasonable to ask if alterations of members of the RAD52 epistasis group lead to tumor development. Here we describe missense mutations at functional regions of RAD54 and the absence of the wild-type RAD54 expression resulting from aberrant splicing in primary cancers. Since RAD54 is a recombinational protein associated with RAD51, this is the first genetic evidence that cancer arises from a defect in repair processes involving homologous recombination.

Allograft transduction of IL-10 prolongs survival following orthotopic liver transplantation.

Interleukin-10 (IL-10) is an ideal candidate cytokine for suppressing the alloimmune response in transplantation. To determine whether genetic modulation of the hepatic graft with IL-10 could prolong survival following orthotopic liver transplantation, we constructed a replication-deficient adenovirus vector expressing human IL-10 (AdCMVhIL-10). Intraportal injection of this vector into a donor rat 24–48 h before grafting resulted in efficient release of IL-10 into the circulation of a recipient rat after transplantation. Moreover, levels of hIL-10 from the suprahepatic vena cava were significantly (1.48-fold) higher than those from the infrahepatic vena cava (P = 0.013), indicating local IL-10 production within the transduced hepatic graft. AdCMVhIL-10 induced a prolongation of median survival to more than 87 days, with two of five transduced grafts showing more than 100 days of ongoing survival, when compared with 11 days for grafts transduced with a control adenovirus vector carrying the E. coli β-galactosidase gene (P = 0.0021) and 11 days for untreated grafts (P = 0.0021). Pathological findings occurring in the AdCMVhIL-10-transduced hepatic grafts revealed no evidence of progressive rejection reaction resulting in graft failure. These results demonstrate that hepatic grafts modulated by IL-10 gene transfer make local and effective immunosuppression feasible in the transplantation setting.