The Tumor Growth Inhibitory Effect of a Standardized Extract of Cultured Lentinula edodes Mycelia Using Patient Derived Xenograft Model.

Patient derived xenograft (PDX) is a powerful tool to confirm pharmacological efficacy in non-clinical studies for the development of various drugs including anti-cancer agents and therapeutic research. A standardized extract of cultured Lentinula edodes mycelia, a product name AHCC® is produced by Amino Up Co., Ltd. (Sapporo, Japan). In this study, we investigated the inhibitory effect of AHCC® on the growth of tumor PDX in Super SCID (severe combined immunodeficiency) mice. Effects of AHCC® and BCG administration on the growth of renal cancer PDX implanted in Super SCID mice were evaluated by PDX growth curve. Tendency for the effects on the growth of renal cancer PDX in Super SCID by administration of AHCC® and BCG before implanting the PDX were demonstrated. The effects of the oral administration of AHCC® on the growth of renal, invasive and non-invasive breast cancer PDX in Super SCID mice were studied. In Super SCID mice transplanted with renal cancer PDX, AHCC® significantly suppressed tumor proliferation from the day 48 to 83 after transplantation. In two types of breast cancer PDX, tendency of the growth inhibitory effects of AHCC® were shown by PDX growth curve. Significant inhibitory effect was found at only one time point for during proliferation in each PDX. Super SCID-PDX model has the potential to be a useful tool to investigate for the effect of functional foods.

Anti-glypican-1 antibody-drug conjugate is a potential therapy against pancreatic cancer.

BACKGROUND: Pancreatic cancer (PDAC) is the most lethal malignancy. New treatment options for it are urgently required. The aim was to develop an antibody-drug conjugate (ADC) targeting glypican-1 (GPC-1) as a new therapy for PDAC. METHODS: We evaluated GPC-1 expression in resected PDAC specimens and PDAC cell lines. We then measured the antitumour effect of anti-GPC-1 monoclonal antibody conjugated with the cytotoxic agent monomethyl auristatin F (MMAF) in vitro and in vivo. RESULTS: GPC-1 was overexpressed in most primary PDAC cells and tissues. The PDAC cell lines BxPC-3 and T3M-4 strongly expressed GPC-1 relative to SUIT-2 cells. Compared with control ADC, GPC-1-ADC showed a potent antitumour effect against BxPC-3 and T3M-4, but little activity against SUIT-2 cells. In the xenograft and patient-derived tumour models, GPC-1-ADC significantly and potently inhibited tumour growth in a dose-dependent manner. GPC-1-ADC-mediated G2/M-phase cell cycle arrest was detected in the tumour tissues of GPC-1-ADC-treated mice relative to those of control-ADC-treated mice. CONCLUSIONS: GPC-1-ADC showed significant tumour growth inhibition against GPC-1-positive pancreatic cell lines and patient-derived, GPC-1-positive pancreatic cancer tissues. Our preclinical data demonstrated that targeting GPC-1 with ADC is a promising therapy for patients with GPC-1-positive pancreatic cancer.

High-throughput screening in colorectal cancer tissue-originated spheroids.

Patient-derived cancer organoid culture is an important live material that reflects clinical heterogeneity. However, the limited amount of organoids available for each case as well as the considerable amount of time and cost to expand in vitro makes it impractical to perform high-throughput drug screening using organoid cultures from multiple patients. Here, we report an advanced system for the high-throughput screening of 2427 drugs using the cancer tissue-originated spheroid (CTOS) method. In this system, we apply the CTOS method in an ex vivo platform from xenograft tumors, using machines to handle CTOS and reagents, and testing a CTOS reference panel of multiple CTOS lines for the hit drugs. CTOS passages in xenograft tumors resulted in minimal changes of morphological and genomic status, and xenograft tumor generation efficiently expanded the number of CTOS to evaluate multiple drugs. Our panel of colorectal cancer CTOS lines exhibited diverse sensitivities to the hit compounds, demonstrating the usefulness of this system for investigating highly heterogeneous disease.

Enhanced fucosylation of GA1 in the digestive tracts of X-ray-irradiated mice.

In mice at 4 days after X-ray-irradiation at 0.5 Gy/min for 16 min, the tissue weights of immune organs, i.e., thymus and spleen, were decreased due to injury to lymphocytes by the X-rays. The resulting immunosuppressive condition allowed the growth of lactobacilli, i.e., L. murinus, which contained LacßTH-DG and possessed the ability to induce transcription of the fucosyltransferase gene for synthesis of FGA1. LacßTH-DG was detected in the jejunal and ileal contents of X-ray-irradiated mice, but not in those of control mice, whereas LacTetH-DG of L. johnsonii was present in the stomach and caecal contents of both mice. The amounts of FGA1 in the duodenal and jejunal tissues of X-ray-irradiated mice increased to 4- and 9-fold of those in controls, respectively. Reflecting the enhanced fucosylation of GA1, the total amounts of FGA1 excreted into the contents of X-ray-irradiated mice were 1.4-times higher than those in controls. Also, when the extent of enhanced fucosylation of GA1 in several regions of the digestive tracts of X-ray-irradiated mice was compared with that in immune deficient nude, scid and pIgR(-/-) mice, the more than 4-fold increases of FGA1 observed in duodenal and jejunal tissues corresponded to those in pIgR(-/-) mice without secretory IgA. Since an increased amount of FGA1 in the small intestine was observed only 4 days after X-ray-irradiation, and diminished synthesis of FGA1 occurred on administration of penicillin and streptomycin, fucosylation of GA1 in the small intestine was revealed to occur quickly in response to a change in the intestinal bacterial population.

p53-Dependent suppression of genome instability in germ cells.

Radiation increases mutation frequencies at tandem repeat loci. Germline mutations in γ-ray-irradiated medaka fish (Oryzias latipes) were studied, focusing on the microsatellite loci. Mismatch-repair genes suppress microsatellite mutation by directly removing altered sequences at the nucleotide level, whereas the p53 gene suppresses genetic alterations by eliminating damaged cells. The contribution of these two defense mechanisms to radiation-induced microsatellite instability was addressed. The spontaneous mutation frequency was significantly higher in msh2(-/-) males than in wild-type fish, whereas there was no difference in the frequency of radiation-induced mutations between msh2(-/-) and wild-type fish. By contrast, irradiated p53(-/-) fish exhibited markedly increased mutation frequencies, whereas their spontaneous mutation frequency was the same as that of wild-type fish. In the spermatogonia of the testis, radiation induced a high level of apoptosis both in wild-type and msh2(-/-) fish, but negligible levels in p53(-/-) fish. The results demonstrate that the msh2 and p53 genes protect genome integrity against spontaneous and radiation-induced mutation by two different pathways: direct removal of mismatches and elimination of damaged cells.

Changes in bacterial glycolipids as an index of intestinal lactobacilli and epithelial glycolipids in the digestive tracts of mice after administration of penicillin and streptomycin.

The major lipid constituent of symbiotic gram-positive bacteria in animals are phosphatidylglycerol, cardiolipin and dihexaosyl diglycerides (DH-DG), whose hydrophobic structures are characteristic of the environments, and the carbohydrate structures of DH-DGs are bacterial species-characteristic. Immunization of rabbits with intestinal lactobacilli generated antibodies against DH-DGs and their modified structures, among which Galα1-6-substituted DH-DG, i.e., Lactobacillus tetrahexaosyl diglyceride (LacTetH-DG), reacted with antibodies more intensely than DH-DG. Whereas, from the 16S-rRNA sequence, the intestinal lactobacilli in murine digestive tracts were revealed to be L. johnsonii, in which LacTetH-DG is present at the concentration of 2.2 ng per 1 × 10(6) cells. To obtain more accurate estimates of intestinal lactobacilli in several regions of the digestive tract of mice, LacTetH-DG was detected by TLC-immunostaining with anti-Lactobacillus antisera, being found in the stomach, cecum and colon of normal breeding mice, 1.0 × 10(9), 3.5 × 10(9) and 7.4 × 10(9) cells, respectively. Administration of penicillin and streptomycin for 6 days resulted in a reduction in the number of intestinal lactobacilli, the levels being 0 %, 30 % and 4 % of the control ones in the stomach, cecum and colon, respectively, which was associated with the accumulation of the contents in the tracts from the stomach to the cecum and with diarrhea. In addition, a reduced amount of fucosyl GA1 (FGA1) and a compensatory increase in GA1 due to the reduced activity of α1,2-fucosyltransferase in the small intestine and the enhanced discharge of FGA1 into the contents occurred in mice, probably due to the altered population of bacteria caused by administration of penicillin and streptomycin.

Bacterial species-characteristic profiles of molecular species, and the antigenicity of phospholipids and glycolipids in symbiotic Lactobacillus, Staphylococcus and Streptococcus species.

Human symbiotic bacteria, Lactobacillus reuteri (LR) in the intestines, Staphylococcus epidermidis (SE) in skin and Streptococcus salivalis (SS) in the oral cavity, contain dihexaosyl diglycerides (DH-DG) in concentrations equivalent to those of phosphatidyl glycerol (PG) and cardiolipin (CL), together with mono- to tetrahexaosyl DGs. The molecular species, as the combination of fatty acids in the DG moiety, were revealed to be bacterial species-characteristic, but to be similar between glycolipids and phospholipids in individual bacteria, the major ones being 16:0 and cy19:0 for LR, ai15:0 and ai17:0 for SE, and 16:0 and 18:1 for SS, respectively. The carbohydrate structures of DH-DGs were also bacterial species-characteristic, being Galα1-2Glcα for LR, Glcß1-6Glcß for SE, and Glcα1-2Glcα for SS, respectively. Also, bacterial glycolipids were revealed to provide antigenic determinants characteristic of bacterial species on immunization of rabbits with the respective bacteria. Anti-L. johnsonii antiserum intensely reacted with tri- and tetrahexaosyl DGs, in which Galα was bound to DH-DG through an α1-6 linkage, as well as with DH-DG from LR. Although anti-SE antiserum preferentially reacted with DH-DG from SE, anti-SS antiserum reacted with DH-DG from SS and, to a lesser extent, with DH-DGs from LR and SE. But, both anti-SE and anti-SS antiserum did not react at all with monohexaosyl DG or glycosphingolipids with the same carbohydrates at the nonreducing terminals. In addition, 75 % of human sera, irrespective of the ABO blood group, were found to contain IgM to tri- and tetrahexaosyl DGs from LR, but not to DH-DGs from LR, SE and SS.

Excretion into feces of asialo GM1 in the murine digestive tract and Lactobacillus johnsonii exhibiting binding ability toward asialo GM1. A possible role of epithelial glycolipids in the discharge of intestinal bacteria.

In the digestive tract of mice (HR-1, 5 months old, ♀), asialo GM1 (GA1) exhibiting receptor activity toward several intestinal bacteria was preferentially expressed in the small intestine. Also, less than 10% of GA1 in the small intestine was converted into fucosylated and sulfated derivatives, but it was completely converted to fucosyl GA1 (FGA1) in the stomach, cecum and colon. Among the lipid components in these tissues, glycolipids other than Forssman antigen and cholesterol sulfate (CS) were present in the digestive tract contents. However, sulfated GA1, sulfatide and fucosyl GM1 in the gastro-intestinal contents were not present in the cecal and colonic contents, in which the major glycolipids were ceramide monohexoside (CMH), GA1 and FGA1. The total amount of GA1 in the whole contents was 20% of that in the tissues. Thus, glycolipids were stable during the process of digestion, and excreted from the body together with cholesterol and CS. On the other hand, Lactobacillus johnsonii (LJ), whose receptor is GA1, was detected in the cecal and colonic contents on sequential analysis of 16S-ribosomal RNA and TLC-immunostaining of antigenic glycolipids with anti-LJ antiserum. LJ was found to comprise 20% of the total bacteria cultured in the lactobacillus medium under aerobic conditions, and to be present in the cecal and colonic contents, 9.8 × 10(7) cells versus 37 µg GA1 and 1.4 × 10(8) cells versus 49 µg GA1, respectively. Thus, GA1 in the contents might facilitate the discharge of intestinal bacteria by becoming attached them to prevent their irregular diffusion.

Glypican-1 Is a Novel Target for Stroma and Tumor Cell Dual-Targeting Antibody-Drug Conjugates in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a stroma-rich cancer. Extracellular matrix proteins produced by cancer-associated fibroblasts (CAFs) found in tumor stroma that impedes effective delivery of chemotherapeutic agents results in poor response in patients with PDAC. Previously, our group reported that glypican-1 (GPC1) was overexpressed in human PDAC and negatively correlated with patient survival. Immunohistochemical analysis of 25 patients with PDAC tumor specimens revealed elevated expression of GPC1 in stromal cells and pancreatic cancer cells in 80% of patients. Interestingly, GPC1 was expressed on CAFs in PDAC. We generated a GPC1 antibody-drug conjugate conjugated with monomethyl auristatin E [GPC1-ADC(MMAE)] and evaluated its preclinical antitumor activity by targeting GPC1-positive CAF and cancer cells in PDAC. GPC1-ADC(MMAE) inhibited the growth of GPC1-positive PDAC cell lines in vitro Furthermore, GPC1-ADC(MMAE) showed a potent antitumor effect in the PDAC patient-derived tumor xenograft (PDX) model against GPC1-positive CAF and heterogeneous GPC1-expressing cancer cells. Notably, GPC1-ADC(MMAE) showed robust preclinical efficacy against GPC1 in a stroma-positive/cancer-negative PDAC PDX model. GPC1-ADC(MMAE) was delivered and internalized to CAFs. Although apoptosis was not observed in CAFs, the released MMAE from CAFs via MDR-1 induced apoptosis of cancer cells neighboring CAFs and efficiently inhibited PDAC tumor growth. GPC1-ADC(MMAE) exhibited potent and unique antitumor activity in GPC1-positive PDAC PDX models, which suggests that GPC1 is a novel therapeutic target in PDAC and other stromal GPC1-positive solid tumors. These findings show that targeting GPC1 on CAF using GPC1-ADC(MMAE) is a useful approach in case of stroma-rich tumors such as PDAC.

A glypican-1-targeted antibody-drug conjugate exhibits potent tumor growth inhibition in glypican-1-positive pancreatic cancer and esophageal squamous cell carcinoma.

An antibody-drug conjugate (ADC) is a promising therapeutic modality because selective and effective delivery of an anti-cancer drug is achieved by drug-conjugated antibody-targeting cancer antigen. Glypican 1 (GPC1) is highly expressed in malignant tumors, including pancreatic ductal adenocarcinoma (PDAC) and esophageal squamous cell carcinoma (ESCC). Herein, we describe the usefulness of GPC1-targeting ADC. Humanized anti-GPC1 antibody (clone T2) was developed and conjugated with monomethyl auristatin E (MMAE) via maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (mc-vc-PABC) linkers (humanized GPC1-ADC[MMAE]). Humanized GPC1-ADC(MMAE) inhibited the growth of GPC1-positive PDAC and ESCC cell lines via inducing cycle arrest in the G2/M phase and apoptosis in vitro. The binding activity of humanized GPC1-ADC(MMAE) with GPC1 was comparable with that of the unconjugated anti-GPC1 antibody. The humanized GPC1-ADC(MMAE) was effective in GPC1-positive BxPC-3 subcutaneously xenografted mice but not in GPC1-negative BxPC-3-GPC1-KO xenografted mice. To assess the bystander killing activity of the humanized GPC1-ADC(MMAE), a mixture of GPC1-positive BxPC-3 and GPC1-negative BxPC-3-GPC1-KO-Luc cells were subcutaneously inoculated, and a heterogenous GPC1-expressing tumor model was developed. The humanized GPC1-ADC(MMAE) inhibited the tumor growth and decreased the luciferase signal, measured with an in vivo imaging system (IVIS), which suggests that the suppression of the BxPC-3-GPC1-KO-Luc population. The humanized GPC1-ADC(MMAE) also inhibited the established liver metastases of BxPC-3 cells and significantly improved the overall survival of the mice. It exhibited a potent antitumor effect on the GPC1-positive PDAC and ESCC patient-derived xenograft (PDX) models. Our preclinical data demonstrate that GPC1 is a promising therapeutic target for ADC.

Identification and Therapeutic Targeting of GPR20, Selectively Expressed in Gastrointestinal Stromal Tumors, with DS-6157a, a First-in-Class Antibody-Drug Conjugate.

Currently, the only approved treatments for gastrointestinal stromal tumor (GIST) are tyrosine kinase inhibitors (TKI), which eventually lead to the development of secondary resistance mutations in KIT or PDGFRA and disease progression. Herein, we identified G protein-coupled receptor 20 (GPR20) as a novel non-tyrosine kinase target in GIST, developed new GPR20 IHC, and assessed GPR20 expression in cell lines, patient-derived xenografts, and clinical samples from two institutes (United States and Japan). We studied GPR20 expression stratified by treatment line, KIT expression, GIST molecular subtype, and primary tumor location. We produced DS-6157a, an anti-GPR20 antibody-drug conjugate with a novel tetrapeptide-based linker and DNA topoisomerase I inhibitor exatecan derivative (DXd). DS-6157a exhibited GPR20 expression-dependent antitumor activity in GIST xenograft models including a GIST model resistant to imatinib, sunitinib, and regorafenib. Preclinical pharmacokinetics and safety profile of DS-6157a support its clinical development as a potential novel GIST therapy in patients who are refractory or have resistance or intolerance to approved TKIs. SIGNIFICANCE: GPR20 is selectively expressed in GIST across all treatment lines, regardless of KIT/PDGFRA genotypes. We generated DS-6157a, a DXd-based antibody-drug conjugate that exhibited antitumor activity in GIST models by a different mode of action than currently approved TKIs, showing favorable pharmacokinetics and safety profiles.This article is highlighted in the In This Issue feature, p. 1307.

Effects of fission neutrons on human thyroid tissues maintained in SCID mice.

Morphology and function (secretion of thyroid hormone) of human thyroid tissues from Graves' disease patients are well maintained in C57BL/6J-scid mice. Serum level of thyroid hormone was reduced by fission neutrons from the nuclear reactor UTR-KINKI, and changes in thyroid hormone by fission neutrons were bigger than those by low LET radiations, X-rays and (137)Cs gamma-rays, suggesting high relative biological effectiveness (RBE; 6.5) of fission neutrons. Microarray analyses revealed that about 3% of genes showed more than 4-fold change in gene expression in the unexposed thyroid tissues against surgically resected thyroid tissues from the same patient, probably due to the difficult oxygen and nutrient supply shortly after transplantation. Dose-dependent changes in gene expression against unexposed concurrent controls were observed with increasing doses of fission neutrons (0.2-0.6Gy) and (137)Cs gamma-rays (1.0-3.0Gy) and showed high RBE (4.2). Furthermore, there were some specific genes which showed more than 4-fold change in gene expression in all the thyroid tissues exposed to higher doses of radiation, especially neutrons (0.4 and 0.6Gy), but none at lower doses (0.2Gy of neutrons and 1.0 and 2.0Gy of gamma-rays). These genes related to degeneration, regeneration, apoptosis, and transcription, respond specifically and very sensitively to neutron injury in human thyroid tissues. This is the first experimental report that fission neutrons can induce some morphological and functional disorders in human tissues, showing high RBE against gamma-ray exposure. These results are useful to evaluate the risks of fission neutrons and cosmic rays to humans.

Spermatogenesis-associated changes of fucosylated glycolipids in murine testis.

By targeted deletion of either the FUT1- or FUT2-gene for α1,2-fucosyltransferase, expression of FGM1 and FGA1, in murine testis was revealed to be sustained through unique interchangeability of the genes, indicating their significant roles for spermatogenesis. Accordingly, we examined the amounts of FGM1 and FGA1 in the testes of mice at 1-42 days after birth in comparison to those of several glycolipids including seminolipid. Although Forssman antigen and GM1 were present in relatively constant amounts during the period examined, GM3, which was the major one at 1 day, quickly decreased during development and had completely disappeared at 4 weeks. The following glycolipids were expressed in stage-specific manners, FGM1 for primary spermatocytes at 1 week, a seminolipid for secondary spermatocytes at 2 weeks, and GM3 lactone and FGA1 for spermatids and spermatozoa at 3 weeks. In fact, immunohistochemical staining with anti-FGM1 and anti-FGA1 antibodies demonstrated that FGM1 and FGA1 were distributed in the spermatocytes, and the spermatids and spermatozoa, respectively, and FGA1, together with seminolipid, were the immunogenic markers of spermatozoa. Thus, the fucosylation of glycolipids is a spermatogenesis-associated event, which should occur even with use of either the FUT1- or FUT2-gene.

Study of glycosylation of prostate-specific antigen secreted by cancer tissue-originated spheroids reveals new candidates for prostate cancer detection.

Prostate-specific antigen (PSA) is the most frequently used biomarker for the screening of prostate cancer. Understanding the structure of cancer-specific glycans can help us improve PSA assay. In the present study, we analysed the glycans of PSA obtained from culture medium containing cancer tissue-originated spheroids (CTOS) which have similar characteristics as that of the parent tumour to explore the new candidates for cancer-related glycoforms of PSA. The glycan profile of PSA from CTOS was determined by comparing with PSA from normal seminal plasma and cancer cell lines (LNCaP and 22Rv1) using lectin chromatography and mass spectrometry. PSA from CTOS was mostly sialylated and the content of Wisteria floribunda agglutinin reactive glycan (LacdiNAc) was similar to that of PSA derived from seminal plasma and 22Rv1. Conversely, concanavalin A (Con A)-unbound PSA was definitely detected from the three cancer origins but was almost negligible in seminal PSA. Two novel types of PSA were elucidated in the Con A-unbound fraction: one is a high molecular weight PSA with highly branched N-glycans, and the other is a low molecular weight PSA without N-glycans. Furthermore, the existence of Lewis X antigen group on PSA was indicated. These PSAs will be candidates for new cancer-related markers.

A Novel HER3-Targeting Antibody-Drug Conjugate, U3-1402, Exhibits Potent Therapeutic Efficacy through the Delivery of Cytotoxic Payload by Efficient Internalization.

PURPOSE: HER3 is a compelling target for cancer treatment; however, no HER3-targeted therapy is currently clinically available. Here, we produced U3-1402, an anti-HER3 antibody-drug conjugate with a topoisomerase I inhibitor exatecan derivative (DXd), and systematically investigated its targeted drug delivery potential and antitumor activity in preclinical models. EXPERIMENTAL DESIGN: In vitro pharmacologic activities and the mechanisms of action of U3-1402 were assessed in several human cancer cell lines. Antitumor activity of U3-1402 was evaluated in xenograft mouse models, including patient-derived xenograft (PDX) models. Safety assessments were also conducted in rats and monkeys. RESULTS: U3-1402 showed HER3-specific binding followed by highly efficient cancer cell internalization. Subsequently, U3-1402 was translocated to the lysosome and released its payload DXd. While U3-1402 was able to inhibit HER3-activated signaling similar to its naked antibody patritumab, the cytotoxic activity of U3-1402 in HER3-expressing cells was predominantly mediated by released DXd through DNA damage and apoptosis induction. In xenograft mouse models, U3-1402 exhibited dose-dependent and HER3-dependent antitumor activity. Furthermore, U3-1402 exerted potent antitumor activity against PDX tumors with HER3 expression. Acceptable toxicity was noted in both rats and monkeys. CONCLUSIONS: U3-1402 demonstrated promising antitumor activity against HER3-expressing tumors with tolerable safety profiles. The activity of U3-1402 was driven by HER3-mediated payload delivery via high internalization into tumor cells.

Transgenerational effects from exposure to environmental toxic substances.

Exposure of mouse germ cells to radiation and chemicals results in mutation, malformation, cancer and other adverse effects (e.g., functional disorders) in the offspring, though these findings have not been proven in human studies. Environmental toxic substances such as urethane (ethyl carbamate) which had been injected subcutaneously to 50 million people as a co-solvent of analgesics and dioxin (an endocrine disruptor) have been found to be associated with adverse effects in the progeny of mice after parental exposures. There are some reports on congenital malformations in the progeny of fathers who had been exposed to dioxin. However, these substances have not shown mutagenicity in in vitro assay systems such as bacterial systems even with S9, cell transformation assays, etc., in spite of their potent teratogenicity and carcinogenicity in in vivo systems. Urethane was negative in the mouse specific locus test for germ cell mutations, but elicited a significant response at the same loci in the offspring of mice treated during pregnancy. Further, urethane is a mutagen in Drosophila germ cell tests, specifically inducing point mutations. Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) does not induce in vivo somatic mutations in mice and rats. It does not induce chromosomal aberrations when the mouse and/or human sperm are treated, but induces mutations at ESTR (expanded simple tandem repeat) loci in mice at low frequencies and also congenital malformations. In this paper, we first present an overview of the results of our studies on transgenerational effects of these toxic substances, compare the results with those obtained after radiation exposure, and then discuss our subsequent studies to reconcile the problems underlying their mutagenicity, teratogenicity and carcinogenicity.

Differential radiation sensitivity to morphological, functional and molecular changes of human thyroid tissues and bone marrow cells maintained in SCID mice.

Morphology and function of human organs and tissues are well maintained in the improved SCID (severe combined immunodeficient) mice for a long period (approximately 3 years). To study the radiation-induced damage on human thyroid gland, human thyroid tissues transplanted to SCID mice were consecutively exposed to X-rays or 137Cs gamma-rays at high and low dose rates for approximately 2 years. Consecutive irradiation resulted in the disappearance of follicles and significant decrease of thyroid hormone secretion. Mutations in p53 and c-kit genes were induced significantly in human thyroid tissues from old head and neck cancer patients (av. 56.8 years, 4 males) and a Graves' disease patient (20 years, male) over the dose of 24 Gy (44.7+/-5.9 Gy, mean+/-S.E) and 11 Gy (20.2+/-7.8 Gy), respectively, while mutations were not detected at lower doses nor in unexposed matched controls (p < 0.01). There were significant differences in mutation frequency in the transplanted human thyroid tissues (31 years, female) between high dose rate (1.19 Gy/min; 8 in 20 tissues) and low dose rate (0.00023 Gy/min; 0 in 14 tissues) exposures (p < 0.01). Mutations were not detected in RET, K-ras and beta-catenin genes. Expression analysis by GeneChip indicated that gene expression was also well maintained in the transplanted human thyroid tissues. However, lower doses (1 or 3 Gy) of 137Cs gamma-rays can induce changes in gene expression in the transplanted human thyroid tissues. Furthermore, fatally irradiated SCID mice could survive with human bone marrow cell transplantation. When about half of mouse bone marrows were replaced by human bone marrow cells, the human bone marrow cells showed high sensitivity to gamma-irradiation; 28.0% and 0.45% survival after 0.5 and 2.0 Gy exposures, respectively.

Distortion of neutron field during mice irradiation at Kinki University reactor UTR-KINKI.

A dosimetry study of mice irradiation at the Kinki University nuclear reactor (UTR-KINKI) has been carried out. Neutron and gamma-ray doses at the irradiation port in the presence of 0, 1, 2, 4 and 6 mice were measured using the paired chamber method. The results show that neutron dose is reduced with increasing numbers of mice. In the six-mice irradiation condition, neutron dose is about 15% smaller compared to a case where no mice were placed in the irradiation port. To investigate the distortion of the neutron spectrum during mice irradiation at UTR-KINKI, a Monte Carlo calculation using the MCNP4C code has been carried out. The measured variation in dose with respect to the total mouse mass was closely reproduced by the calculation results for neutron and gamma-ray dose. Distortion of the neutron spectrum was observed to occur between 1 keV and 1 MeV.

Gene mutation analysis of sinonasal lymphomas in Indonesia.

Sinonasal lymphomas comprise NK/T-cell (NKTCL) type and B-cell type with unique geographical development. In this study, mutations of p53, K-ras, c-kit, beta-catenin, and bak gene were analyzed using polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) followed by direct sequencing in 41 sinonasal lymphomas (27 NKTCL and 14 B-cell type) from Indonesia. In situ hybridization study with EBER-1 probe revealed that 85% of NKTCL cases were EBV positive, but none of B-cell type was EBV positive. Frequency of mutations in p53, K-ras, c-kit, beta-catenin, and bak gene was 62.9%, 0%, 11.1%, 18.5%, and 25.9%, respectively, in NKTCL, and 71.4%, 0%, 23.1%, 21.4%, and 57.1%, respectively, in B-cell cases, showing that mutation frequency in all genes was higher in B-cell than in NKTCL cases. These findings suggest that gene mutations might be the driving-force for B-cell lymphoma, whereas combined EBV infection and gene mutations contribute to NKTCL development in Indonesia.

Neutron relative biological effectiveness in Hiroshima and Nagasaki atomic bomb survivors: a critical review.

The calculated risk of cancer in humans due to radiation exposure is based primarily on long-term follow-up studies, e.g. the life-span study (LSS) on atomic bomb (A-bomb) survivors in Hiroshima and Nagasaki. Since A-bomb radiation consists of a mixture of γ-rays and neutrons, it is essential that the relative biological effectiveness (RBE) of neutrons is adequately evaluated if a study is to serve as a reference for cancer risk. However, the relatively small neutron component hampered the direct estimation of RBE in LSS data. To circumvent this problem, several strategies have been attempted, including dose-independent constant RBE, dose-dependent variable RBE, and dependence on the degrees of dominance of intermingled γ-rays. By surveying the available literature, we tested the chromosomal RBE of neutrons as the biological endpoint for its equivalence to the microdosimetric quantities obtained using a tissue-equivalent proportional counter (TEPC) in various neutron fields. The radiation weighting factor, or quality factor, Qn, of neutrons as expressed in terms of the energy dependence of the maximum RBE, RBEm, was consistent with that predicted by the TEPC data, indicating that the chromosomally measured RBE was independent of the magnitude of coexisting γ-rays. The obtained neutron RBE, which varied with neutron dose, was confirmed to be the most adequate RBE system in terms of agreement with the cancer incidence in A-bomb survivors, using chromosome aberrations as surrogate markers. With this RBE system, the cancer risk in A-bomb survivors as expressed in unit dose of reference radiation is equally compatible with Hiroshima and Nagasaki cities, and may be potentially applicable in other cases of human radiation exposure.