Enhanced efficacy of the novel recombinant clone VasSF in a mouse model of antineutrophil cytoplasmic antibody-associated vasculitis.

Based on the efficacy of intravenous immunoglobulin (IVIg) for the treatment of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), we developed a recombinant single-chain-fragment variable clone, VasSF, therapeutic against AAV in a mouse model (SCG/Kj mice). VasSF is thought to bind to vasculitis-associated apolipoprotein A-II (APOA2) as a target molecule. VasSF is a promising new drug against AAV, but difficulties in the yield and purification of VasSF remain unresolved. We produced monomers of new VasSF molecules by modifying the plasmid structure for VasSF expression and simplifying the purification method using high-performance liquid chromatography. We compared the therapeutic effects between 5-day continuous administration of the monomers, as in IVIg treatment, and single shots of 5-day-equivalent doses. We also evaluated the life-prolonging effect of the single-shot treatment. Two-dimensional western blots were used to examine the binding of VasSF to APOA2. Our improved manufacturing method resulted in a 100-fold higher yield of VasSF than in our previous study. Monomerization of VasSF stabilized its efficacy. Single shots of a small amount (1/80 000 of IVIg) produced sufficient therapeutic effects, including decreased glomerular crescent formation, a decreasing trend of serum ANCA against myeloperoxidase (MPO-ANCA), decreases in multiple proinflammatory cytokines, and a trend toward prolonged survival. Two-dimensional western blots confirmed the binding of VasSF to APOA2. The newly produced pure VasSF monomers are stable and therapeutic for AAV with a single low-dose injection, possibly by removing vasculitis-associated APOA2. Thus, the new VasSF described herein is a promising drug against AAV.

Trends in Prescribing Antipsychotics for Children and Adolescents in Japan: A Descriptive Epidemiological Study Using a Large-Scale Pharmacy Dataset.

Little is known about antipsychotic prescription patterns among children and adolescents in Japan, particularly in outpatient settings. We investigated the prevalence and trends of antipsychotic prescription for outpatients aged ≤ 17 years receiving a first antipsychotic prescription from 2006 to 2012 based on a large-scale dispensation dataset. Measurements included age, sex, department of diagnosis and treatment, type of prescription (monotherapy or polytherapy), antipsychotic dosage, and concomitant psychotropic drugs. Of the 10,511 patients, 65.1% were aged 13-17 years, and 52.9% were males. Second-generation antipsychotic monotherapy prescriptions increased from 53.8% in 2006 to 78.3% in 2012. Risperidone was the most frequently prescribed antipsychotic, followed by aripiprazole and olanzapine. Approximately 25.0% of patients were prescribed an initial dose less than recommended. Second-generation antipsychotic monotherapy is currently the most frequent prescription pattern among outpatients aged ≤ 17 years receiving an initial antipsychotic prescription.

Effects of administering different vehicles via single intratracheal instillation on responses in the lung and pleural cavity of Crl:CD(SD) rats.

Intratracheal instillation is the introduction of a substance directly into the trachea. Intratracheal instillation has been used to investigate the lung toxicity of several chemicals and requires the suspension or dissolution of test material in a vehicle for even dispersal throughout the lung. Importantly, the toxicities of vehicles used in intratracheal instillation studies are generally considered to be insignificant. Hence, evaluating the influence of different vehicles on the lung due to intratracheal instillation is crucial. We examined the toxic effects of pure water, saline, phosphate buffered saline (PBS), 0.5% Kolliphor® P188 (KP188), 0.1% Tween 20 in saline, and 1.0% BSA in PBS. These vehicles were administered to male Crl:CD(SD) rats by a single intratracheal instillation. On day 3, broncho-alveolar lavage fluid (BALF) from the right lung was collected and processed for cell counting and biochemical analysis, while the left lung was used for histopathological examination. Accumulation of alveolar macrophages was observed in all vehicle-treated groups but was minimal in the group administered saline, somewhat higher in the groups administered pure water, PBS, 0.1% Tween 20, and 1% BSA, and notably higher in the group administered 0.5% KP188. The results from BALF analysis indicated that intratracheal instillation of 0.5% KP188 also induced alveolar damage. Additionally, administering pure water did not appear to cause tissue damage. Eosinophil infiltration in the interstitial regions was histopathologically observed. Altogether, the results of this study are helpful for the selection of appropriate vehicles for use in intratracheal instillation studies.

Adherence and Concomitant Medication Use among Patients on Warfarin Therapy: Insight from a Large Pharmacy Dispensing Database in Japan.

Warfarin is a drug used for anticoagulation management, with a narrow therapeutic range and multiple drug-drug interactions. Adherence and proper use of concomitant medication are thus fundamental to the efficacy and safety of warfarin therapy. In 2012, we retrospectively analyzed data from three large-scale pharmacy chains in Japan. We included all adults (≥ 20 years old) with at least one record of warfarin dispensation. We examined patient demographic data, adherence as measured by medication possession ratio (MPR), and co-dispensation focusing on the number of concomitant dispensations and concurrent use of medications that increase bleeding risk. Thresholds of underadherence and overadherence were set at <0.9 and >1.1, considering the narrow therapeutic window. We reviewed 443007 warfarin dispensation records of 71340 individuals (median age, 73 years; 62% male). The MPR was 1.0 (interquartile range: 0.96-1.0), and underadherence and overadherence was found in 16.3 and 1.9% of individuals, respectively. The median number of co-dispensed drugs was eight at each pharmacy encounter, which did not differ by age group. Drugs associated with a high bleeding risk were dispensed in 40.0% of encounters and accounted for 16.4% of all co-dispensed drugs. In summary, we found optimal overall adherence, as assessed by MPR, among our Japanese study population, even when defining a strict cut-off value. However, polypharmacy was common in all age groups and medications with a high bleeding risk profile were often co-dispensed with warfarin. Future research addressing how these dispensation patterns affect patient outcome is warranted.

Carcinogenicity of intermediate frequency magnetic field in Tg.rasH2 mice.

Although the likelihood of exposure to leaking intermediate frequency magnetic fields (MFs) from electronic devices, such as induction-heating and wireless power transfer systems, has increased, biological data assessing the health risks associated with human exposure remain insufficient. We examined the carcinogenicity of a 20 kHz MF, a typical frequency produced by induction-heating cookers, using a transgenic rasH2 mouse model. Twenty-five male and female CByB6F1-Tg(HRAS)2Jic mice were exposed to a 0.20 mT, 20 kHz MF (22 h/day) or sham-exposed for 26 weeks. As a positive control, 10 male and female rasH2 mice from the same batch were administered a single intraperitoneal injection of 75 mg/kg N-methyl-N-nitrosourea. A blinded histopathological evaluation was performed, and the same experiments were conducted twice, independently, to confirm the reproducibility of the results. Histopathological examination revealed that spontaneous neoplastic lesions, such as splenic hemangiosarcomas and gastric squamous cell papillomas, were less (1-3 per group) in the MF- and sham-exposed groups. The frequency of the neoplastic lesions was not significantly different between the groups. Eight to ten mice in each positive-control group exhibited malignant lymphoma. The outcomes were consistent between duplicated experiments, which indicates lack of carcinogenicity of 20 kHz MF in the rasH2 mouse model. Bioelectromagnetics. © 2019 The Authors. Bioelectromagnetics Published by Wiley Periodicals, Inc.

Prescription of Antidementia Drugs and Antipsychotics for Elderly Patients in Japan: A Descriptive Study Using Pharmacy Prescription Data.

BACKGROUND: Antipsychotics are commonly used for managing behavioral and psychological symptoms of dementia among elderly patients with dementia receiving antidementia drugs (ADDs). However, the use of antipsychotics among these patients has not been investigated since 3 ADDs were approved in 2011 in Japan. METHOD: We conducted a descriptive study using pharmacy prescription data and identified patients aged ≥65 years who were newly prescribed donepezil, memantine, rivastigmine, and galantamine between January 1, 2012, and September 30, 2014. We determined the proportion of antipsychotic prescription and the factors affecting antipsychotic prescription using multivariable Cox proportional hazard models. RESULT: Of 13 876 patients, 1705 were memantine users, and the proportion of antipsychotic prescription among them was the highest (11.1%). Adjusted hazard ratios for donepezil, rivastigmine, and galantamine were 0.66, 0.56, and 0.66, respectively, relative to that for memantine. CONCLUSION: Compared to other ADD users, new memantine users were most likely to be prescribed antipsychotics.

Lung Tumor Induction by 26-week Dermal Application of 1,2-Dichloroethane in CB6F1-Tg rasH2 Mice.

Short-term alternatives to traditional 2-year carcinogenic studies in rodents are being actively pursued. Recently, a 26-week short-term carcinogenicity study using CB6F1-Tg rasH2@Jcl (rasH2) mice has become a worldwide standard for the evaluation of chemical carcinogenesis. However, an acceptable short-term carcinogenic study model for dermally applied products is still lacking. To investigate the suitability of using the rasH2 mouse to test carcinogenic potential, 1,2-dichloroethane (1,2-DCE) was dermally applied to rasH2 mice: 1,2-DCE is a known carcinogen that causes lung bronchiolo-alveolar adenomas and adenocarcinomas when administered topically, orally, or by inhalation exposure; 1,2-DCE at a dose level of 126 mg/mouse in 200 µl acetone or acetone alone (vehicle control) was applied to the dorsal skin of 10 mice of each sex 3 times a week for 26 weeks. As a positive control, 10 mice of each sex received a single intraperitoneal injection of 75 mg/kg of N-methyl- N-nitrosourea. Bronchiolo-alveolar adenomas and adenocarcinomas were significantly increased in 1,2-DCE-treated rasH2 mice of both sexes, and bronchiolo-alveolar hyperplasias were significantly increased in female mice. Overall, almost all mice of each sex developed adenomas and/or adenocarcinomas with 100% of female rasH2 mice developing bronchiolo-alveolar adenocarcinomas.

Trends in antipsychotic prescriptions for Japanese outpatients during 2006-2012: a descriptive epidemiological study.

PURPOSE: This study aimed to assess the trends in antipsychotic prescriptions for outpatients in Japan, where a community-based approach to mental healthcare is emphasized. METHODS: This descriptive epidemiological study used claims data from 1038 community pharmacies across Japan. Outpatients who were ≥18 years old and receiving their initial antipsychotic prescription during 2006-2012 were evaluated. The annual trends were reported for monotherapies, polypharmacy, antipsychotic doses, and the concurrent prescription of psychotropic medications. RESULTS: The 152 592 outpatients included 101 133 (66%) adults (18-64 years old) and 51 459 (34%) older adults (≥65 years old). Among the adults, second-generation antipsychotic monotherapy prescriptions increased from 49% in 2006 to 71% in 2012, first-generation antipsychotic monotherapy prescriptions decreased from 29 to 14%, and antipsychotic polypharmacy decreased from 23 to 15%, respectively. Among the older adults, second-generation antipsychotic monotherapy prescriptions increased from 64 to 82%, first-generation antipsychotic monotherapy prescriptions decreased from 29 to 12%, and antipsychotic polypharmacy decreased from 7 to 6%, respectively. During the study period, >80% of the adults and >90% of the older adults received antipsychotics at risperidone-equivalent doses of <6 mg/day. Anxiolytics/hypnotics, antidepressants, antiparkinson agents, mood stabilizers, and anti-dementia agents were concurrently prescribed with antipsychotics for 70, 33, 20, 20, and 0.3% of the adults and for 43, 16, 19, 8, and 16% of the older adults, respectively. CONCLUSIONS: The present study evaluated large-scale claims-based datasets and found that high-dose prescriptions and antipsychotic polypharmacy among Japanese outpatients were not as prevalent as has been previously thought. Copyright © 2017 John Wiley & Sons, Ltd.

Promotion of liver and kidney carcinogenesis by ethyl tertiary-butyl ether (ETBE) in male Wistar rats.

Tumor-promoting effects of ethyl tertiary-butyl ether (ETBE) were investigated in a 2-stage carcinogenesis bioassay with regard to hepatic and renal carcinogenesis in rats. Male 6-week-old Wistar rats were given drinking water containing N-ethyl-N-(2-hydroxyethyl)nitrosamine (EHEN), as an initiator, at a dose of 500 ppm for 2 weeks. Starting one week thereafter, the animals were administered ETBE at dose levels of 0 (control), 100, 300, 500 or 1,000 mg/kg/day by gavage for 19 weeks from week 4 to 22. Necropsy of all rats was performed at week 23, and livers and kidneys were examined histopathologically. Incidences of hepatocellular adenomas, and those of combined hepatocellular adenomas and carcinomas were significantly elevated in rats given 1,000 mg/kg/day ETBE, but not 100‒500 mg/kg/day ETBE, and there was a significant increase in the average numbers of lesions. No significant differences in incidences and average numbers of renal tubule neoplasms were found in rats administered 100‒1,000 mg/kg/day ETBE. However, the average numbers of atypical tubule hyperplasias, considered to be preneoplastic lesions, were significantly increased in rats given ETBE at 1,000 mg/kg/day, but not in rats given 500 mg/kg/day or lower doses. Thus, these results imply that ETBE has hepatic and renal tumor-promoting activities that affect EHEN-induced carcinogenesis in male rats, and the no-observed-effect level is 500 mg/kg/day under the present experimental conditions.

Tumor necrosis factor-α promotes bile ductular transdifferentiation of mature rat hepatocytes in vitro.

We previously showed that mature hepatocytes could transdifferentiate into bile ductular cells when placed in a collagen-rich microenvironment. To explore the mechanism of transdifferentiation, we examined whether inflammatory cytokines affected the phenotype of hepatocytes in a three-dimensional culture system. Spheroidal aggregates of rat hepatocytes were embedded within a type I collagen gel matrix and cultured in the presence of various cytokines. In the control, hepatocytes gradually lost expression of albumin, tyrosine aminotransferase, and hepatocyte nuclear factor (HNF)-4α, while aberrantly expressed bile ductular markers, including cytokeratin 19 (CK 19) and spermatogenic immunoglobulin superfamily (SgIGSF). Among the cytokines examined, tumor necrosis factor (TNF)-α inhibited expression of albumin and HNF-4α, both at mRNA and protein levels. After culturing for 2 weeks with TNF-α, hepatocytic spheroids were transformed into extensively branching tubular structures composed of CK 19- and SgIGSF-positive small cuboidal cells. These cells responded to secretin with an increase in secretion and expressed functional bile duct markers. TNF-α also induced the phosphorylation of Jun N-terminal kinase (JNK) and c-Jun, and the morphogenesis was inhibited by SP600125, a specific JNK inhibitor. Furthermore, in chronic rat liver injury induced by CCl(4) , ductular reaction in the centrilobular area demonstrated strong nuclear staining of phosphorylated c-Jun. Our results demonstrate that TNF-α promotes the ductular transdifferentiation of hepatocytes and suggest a role of TNF-α in the pathogenesis of ductular reaction.

Recovery of mature hepatocytic phenotype following bile ductular transdifferentiation of rat hepatocytes in vitro.

We previously demonstrated that mature rat hepatocytes transdifferentiate to bile ductular cells when cultured in a three-dimensional collagen-rich matrix. Here, we show that the phenotype of transdifferentiated hepatocytes can be reversed by modulating culture conditions. Spheroidal aggregates of hepatocytes were cultured within a collagen gel matrix in the presence of serum and tumor necrosis factor-α. Spheroids transformed into ductular structures composed of small cuboidal cells, lost the expression of hepatocytic markers, whereas aberrantly expressed bile ductular markers. The transdifferentiated cells were then retrieved from the gels, plated on surfaces coated with a basement membrane-like material, and cultured in serum-free media. Cells spontaneously formed spheroidal aggregates and recovered hepatocytic phenotype. Dexamethasone (Dex), which suppressed the phosphorylation of ERK and Jun N-terminal kinase, facilitated the recovery, and the combination with interleukin-6 or oncostatin M resulted in the recovery of hepatocyte nuclear factor 4 α protein expression and the typical hepatocytic morphology, and a decrease in the expression of bile ductular markers. A cDNA microarray analysis revealed that the hepatocyte-specific mRNA expression profile was recovered in these cells. Our results demonstrate that hepatocytes are able to recover their phenotypes following bile ductular transdifferentiation, suggesting that hepatocytic and bile ductular phenotypes may be mutually reversible.

No Promoting Effect of Ethyl Tertiary-butyl Ether (ETBE) on Rat Urinary Bladder Carcinogenesis Initiated with N-Butyl-N-(4-hydroxybutyl)nitrosamine.

The effects of ethyl tertiary-butyl ether (ETBE) on two-stage urinary bladder carcinogenesis in male F344 rats initiated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) were investigated at various dose levels with regard to possible promoting activity. Groups of 30 rats were given drinking water containing 500 ppm BBN, as an initiator, for 4 weeks and starting one week thereafter received ETBE by gavage (daily, 7 days/week) at dose levels of 0 (control), 100, 300, 500 or 1000 mg/kg/day until experimental week 36. No statistically significant differences in incidences of preneoplastic lesions, papillomas, and carcinomas of the urinary bladder were evident in rats treated with 100-1000 mg/kg/day ETBE as compared with control values. Furthermore, the average numbers of preneoplastic or neoplastic lesions per unit length of basement membrane in rats given 100-1000 mg/kg/day ETBE were also comparable to control values. However, papillomatosis of the urinary bladder was found in 4 out of 30 rats (13%) in the group given 1000 mg/kg/day ETBE, and soft stones in the urinary bladder were found in 3 out of these 4 rats. The results thus demonstrated that ETBE did not exert promotional activity on urinary bladder carcinogenesis. However, papillomatosis of the urinary bladder developed in small numbers of the rats given ETBE at 1000 mg/kg/day but not in rats given 500 mg/kg/day or lower doses.

Calcium signal regulates temperature-dependent transformation of sporozoites in malaria parasite development.

The infection by the malaria parasite of its mammalian host is initiated by the asexual reproduction of the parasite within the host hepatocyte. Before the reproduction, the elongated sporozoites undergo a depolarizing morphogenesis to the spherical exo-erythrocytic form (EEF). This change can be induced in vitro by shifting the environmental conditions, in the absence of host hepatocytes. Using rodent malaria parasites expressing a FRET-based calcium sensor, YC3.60, we observed that the intracellular calcium increased at the center of the bulbous structure during sporozoite transformation. Modulators of intracellular calcium signaling (A23187 and W-7) accelerated the sporozoite-rounding process. These data suggest that calcium signaling regulates the morphological development of the malaria parasite sporozoite to the EEF, and support a fundamental role for calcium as a universal transducer of external stimuli in the parasitic life cycle.

Enhancement of Photosynthetic Iron-Use Efficiency Is an Important Trait of Hordeum vulgare for Adaptation of Photosystems to Iron Deficiency.

Leaf iron (Fe) contents in Fe-deficiency-tolerant plants are not necessarily higher than that in Fe-deficiency-susceptible ones, suggesting an unknown mechanism involved in saving and allowing the efficient use of minimal Fe. To quantitatively evaluate the difference in Fe economy for photosynthesis, we compared the ratio of CO2 assimilation rate to Fe content in newly developed leaves as a novel index of photosynthetic iron-use efficiency (PIUE) among 23 different barley (Hordeum vulgare L.) varieties. Notably, varieties originating from areas with alkaline soil increased PIUE in response to Fe-deficiency, suggesting that PIUE enhancement is a crucial and genetically inherent trait for acclimation to Fe-deficient environments. Multivariate analyses revealed that the ability to increase PIUE was correlated with photochemical quenching (qP), which is a coefficient of light energy used in photosynthesis. Nevertheless, the maximal quantum yield of photosystem II (PSII) photochemistry, non-photochemical quenching, and quantum yield of carbon assimilation showed a relatively low correlation with PIUE. This result suggests that the ability of Fe-deficiency-tolerant varieties of barley to increase PIUE is related to optimizing the electron flow downstream of PSII, including cytochrome b6f and photosystem I.

Significance of integrin αvß5 and erbB3 in enhanced cell migration and liver metastasis of colon carcinomas stimulated by hepatocyte-derived heregulin.

To study the mechanisms of the highly liver-metastatic character of colon carcinoma cells, we studied the expression pattern of surface integrins on LS-LM6 (a highly liver-metastatic human colon cancer cell line) and the effects of hepatocyte-derived soluble factors on cell migration. LS-LM6 showed significantly higher expression of integrin αvß5, a ligand for vitronectin (VN), as compared with its parental cell line (LS174T). A conditioned medium of cultured mouse hepatocytes enhanced VN-mediated cell migration of LS-LM6, which was blocked by neutralizing antibody against integrin αvß5, while the medium did not affect cell adhesion to VN-coated plastic surfaces. The conditioned medium induced phosphorylation of erbB3 and its heterodimeric partner, erbB2. Heregulin (HRG), a ligand for erbB3, exerted similar effects on VN-mediated cell migration and phosphorylation of erbB3 and erbB2. The conditioned medium contained HRG, and depletion of HRG from the medium by pre-absorption with HRG antibody abolished its effects on cell migration. Heregulin (HRG) was expressed in some hepatocytes in the liver with carcinoma cell metastasis. Furthermore, knockdown of integrin αv and erbB3 by small-interfering RNAs significantly inhibited cell migration induced by HRG as well as liver metastasis in vivo. Finally, we found that HRG-induced cell migration was associated with marked phosphorylation of Akt and that cell migration was suppressed by treatment with specific inhibitors of phosphatidylinositol 3-kinase. Our study suggests that hepatocyte-derived HRG might participate in a highly liver-metastatic phenotype of LS-LM6 through enhancement of integrin αvß5-mediated cell migration and erbB3/erbB2 signaling.

ADF2 is required for transformation of the ookinete and sporozoite in malaria parasite development.

Malaria parasites undergo two rounding-up transformations in their life cycle: the ookinete-to-oocyst transformation in the mosquito midgut, and the sporozoite-to-EEF (exo-erythrocytic form) differentiation in the host hepatocyte. Both events are characterized by the loss of polarity, implying that cytoskeletal reorganization is involved. In other eukaryotes, regulation of the actin skeleton is fundamental to subcellular remodeling. Although filamentous actin is well known to be involved in the motility of apicomplexan parasites, its participation in their morphological regulation is still largely unexplored. Here we describe the fundamental role of Actin depolymerization factor 2 (ADF2), a vector-stage-specific ADF isoform, in morphological changes accompanying the parasite life cycle. Among protozoan parasites, Plasmodium is unique in having two actin and two ADF genes. Disruption of the ADF2 gene in the rodent malaria parasite P. berghei had no effect on ookinete development or its subsequent invasion of the midgut. However, both the ookinete-to-oocyst and sporozoite-to-EEF transformations showed significant defects. These results indicated that Plasmodium ADF2 plays a pivotal role in transformation in the malaria parasite life cycle.

Suppressive effect of orthovanadate on hepatic stellate cell activation and liver fibrosis in rats.

Orthovanadate (OV), an inhibitor of protein tyrosine phosphatases, affects various biological processes in a cell-type-specific manner. In this study, we investigated the effect of OV on hepatic stellate cells (HSCs). When primary rat HSCs were cultured in the presence of 10% serum, they spontaneously lost characteristic stellate morphology, proliferated, and were transformed into an activated state with the formation of abundant stress fibers and increased expression of both alpha-smooth muscle actin and collagen type I mRNA. OV treatment inhibited proliferation and activation of HSCs and partially reversed the phenotype of activated HSCs. Among the signaling molecules investigated, phosphorylation of the Src protein at tyrosine 416 was the most striking in OV-treated HSCs. Treatment of cells with Src family inhibitors partially abrogated the effects of OV. Furthermore, transfection of v-Src into activated HSCs induced a stellate morphology similar to that in the quiescent state. We then examined whether OV could effectively suppress HSC activation in vivo after liver injury induced by either carbon tetrachloride or dimethylnitrosamine. OV significantly reduced the appearance of alpha-smooth muscle actin-positive cells and decreased collagen deposition, concomitant with an improvement in liver function. Our study showed for the first time that OV was able to suppress the activation of HSCs, possibly through the modulation of Src activity, and attenuated fibrosis after chronic liver injury.

High human T cell leukemia virus type-1(HTLV-1) provirus load in patients with HTLV-1 carriers complicated with HTLV-1-unrelated disorders.

BACKGROUND: To address the clinical and virological significance of a high HTLV-1 proviral load (VL) in practical blood samples from asymptomatic and symptomatic carriers, we simultaneously examined VL and clonal expansion status using polymerase chain reaction (PCR) quantification (infected cell % of peripheral mononuclear cells) and Southern blotting hybridization (SBH) methods. RESULTS: The present study disclosed extremely high VL with highly dense smears with or without oligoclonal bands in SBH. A high VL of 10% or more was observed in 16 (43.2%) of a total of 33 samples (one of 13 asymptomatic carriers, 8 of 12 symptomatic carriers, and 7 of 8 patients with lymphoma-type ATL without circulating ATL cells). In particular, an extremely high VL of 50% or more was limited to symptomatic carriers whose band findings always contained at least dense smears derived from polyclonally expanded cells infected with HTLV-1. Sequential samples revealed that the VL value was synchronized with the presence or absence of dense smears, and declined at the same time as disappearing dense smears. Dense smears transiently emerged at the active stage of the underlying disease. After disappearance of the smears, several clonal bands became visible and were persistently retained, explaining the process by which the clonality of HTLV-1-infected cells is established. The cases with only oligoclonal bands tended to maintain a stable VL of around 20% for a long time. Two of such cases developed ATL 4 and 3.5 years later, suggesting that a high VL with oligoclonal bands may be a predisposing risk to ATL. CONCLUSION: The main contributor to extremely high VL seems to be transient emergence of dense smears detected by the sensitivity level of SBH, corresponding to polyclonal expansion of HTLV-1-infected cells including abundant small clones. Major clones retained after disappearance of dense smears stably persist and acquire various malignant characteristics step by step.

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)-induced hepatocarcinogenesis is not enhanced by CYP1A inducers, alpha- and beta-naphthoflavone: relationship to intralobular distribution of CYP1A expression.

Interaction of more than two chemicals from foods is a very important factor for carcinogenic risk assessment and management. 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), one of the most abundant carcinogenic heterocyclic amines in cooked foods, is speculated to be a human liver carcinogen. MeIQx is metabolically activated by CYP1A2 and then N-acetyltransferase (NAT), findings that suggest that its carcinogenic potential might be enhanced by simultaneous exposure to chemical(s) inducing CYP1A2. Therefore, we here investigated the effects of alpha- and beta-naphthoflavone as CYP1A2 inducers on MeIQx-induced rat hepatocarcinogenesis in a medium-term rat liver bioassay. Unexpectedly, no modifying influence of naphthoflavones on MeIQx-induced hepatocarcinogenesis was demonstrated with reference to glutathione S-transferase placental form (GST-P) positive foci in the liver, although up-regulation of CYP1A2 was detected on Western blot analysis. Activity of NAT was not affected. In MeIQx-treated rats, CYP1A expression was mainly detected in zone 3 of the liver where GST-P positive foci were preferentially located, while naphthoflavones alone or combinations of naphthoflavones and MeIQx induced CYP1A expression in zone 1. This difference in intralobular distribution of CYP1A might be related to the fact that MeIQx hepatocarcinogenesis was not modified by the two CYP1A inducers.

Maternal exposure to anti-androgenic compounds, vinclozolin, flutamide and procymidone, has no effects on spermatogenesis and DNA methylation in male rats of subsequent generations.

To verify whether anti-androgens cause transgenerational effects on spermatogenesis and DNA methylation in rats, gravid Crl:CD(SD) female rats (4 or 5/group, gestational day (GD) 0=day sperm detected) were intraperitoneally treated with anti-androgenic compounds, such as vinclozolin (100 mg/kg/day), procymidone (100 mg/kg/day), or flutamide (10 mg/kg/day), from GD 8 to GD 15. Testes were collected from F1 male pups at postnatal day (PND) 6 for DNA methylation analysis of the region (210 bp including 7 CpG sites) within the lysophospholipase gene by bisulfite DNA sequencing method. F0 and F1 males underwent the sperm analysis (count, motility and morphology), followed by DNA methylation analysis of the sperm. Remaining F1 males were cohabited with untreated-females to obtain F2 male pups for subsequent DNA methylation analysis of the testes at PND 6. These analyses showed no effects on spermatogenesis and fertility in F1 males of any treatment group. DNA methylation status in testes (F1 and F2 pups at PND 6) or sperms (F1 males at 13 weeks old) of the treatment groups were comparable to the control at all observation points, although DNA methylation rates in testes were slightly lower than those in sperm. In F0 males, no abnormalities in the spermatogenesis, fertility and DNA methylation status of sperm were observed. No transgenerational abnormalities of spermatogenesis and DNA methylation status caused by anti-androgenic compounds were observed.