Clinical and ultrasound features of difficult-to-treat rheumatoid arthritis: A multicenter RA ultrasound cohort study.

OBJECTIVE: The optimal strategy for difficult-to-treat (D2T) rheumatoid arthritis (RA) has not been identified, and the ultrasound characteristics of D2T RA have not been reported. We investigated the clinical characteristics and factors contributing to the outcome in D2T RA in a multicentre RA ultrasound observational cohort. METHOD: We reviewed 307 Japanese patients diagnosed with RA who underwent treatment with biological and targeted synthetic disease-modifying anti-rheumatic drugs (b/tsDMARDs). We compared the differences in patient characteristics between the D2T RA and non-D2T RA groups. We examined the factors contributing to a good response [defined as b/tsDMARD continuation and Clinical Disease Activity Index (CDAI) ≤ 10 at 12 months] in the D2T RA patient group. RESULTS: Forty-three patients (14%) were categorized as D2T RA and the remaining 264 (86%) as non-D2T RA at baseline. The grey-scale (GS) score, disease duration, and CDAI at the initiation of treatment were significantly higher in the D2T RA group than in the non-D2T RA group. In contrast, the power Doppler (PD) score was not significantly different between the two groups. Of the 43 D2T RA patients, 20 achieved a good response. The introduction of CTLA4-Ig (n = 5) was significantly associated with a good response in analysis based on inverse probability weighting with propensity score. GS and PD scores at baseline were not significantly associated with therapeutic response at 12 months in D2T RA patients. CONCLUSIONS: Patients with D2T RA had high clinical and ultrasound activity and poor responses to treatment with b/tsDMARDs. CTLA4-Ig was associated with a good response at 12 months in D2T RA patients.

Ultrasound efficacy of targeted-synthetic disease-modifying anti-rheumatic drug treatment in rheumatoid arthritis: a multicenter prospective cohort study in Japan.

OBJECTIVE: This study investigated the effectiveness of treatment with Janus kinase (JAK) inhibitors in rheumatoid arthritis (RA) assessed by ultrasonography (US) activity, and the influence of patient characteristics and previous treatments. METHOD: This prospective study assessed 60 treatment initiations among 53 Japanese patients diagnosed with RA who underwent treatment with JAK inhibitors during June 2013 to February 2020. Of the 53 patients, seven patients were enrolled in duplicate because they were treated with two different JAK inhibitors at different periods. For each case, the improvement rate on the power Doppler (PD) score was assessed at 6 month follow-up. Median improvement rate of PD score was used to classify cases as either US responders or non-responders, and patient characteristics were compared between the two groups. RESULTS: All indicators of clinical disease activity and US activity showed a significant improvement at 3 months compared with baseline. Although the JAK inhibitor-cycler group and the interleukin-6 (IL-6) inhibitor inadequate response (IR) group tended to show a later improvement for US activity, all indicators of clinical disease activity and US activity showed a significant improvement at 6 months compared with baseline for both groups. Multivariate analysis showed that concomitant methotrexate use and an IR to the previous biologic or targeted-synthetic disease-modifying anti-rheumatic drug (b/tsDMARD) treatment were independently and significantly associated with US responders. CONCLUSION: Use of a JAK inhibitor in combination with methotrexate and an absence of IR to any previous b/tsDMARDs demonstrated superior effectiveness for patients with RA.

Significance of anti-Ro/SSA antibodies in the response and retention of abatacept in patients with rheumatoid arthritis: a multicentre cohort study.

Objective: To determine whether the positivity of baseline anti-Ro/Sjögren's syndrome antigen A (SSA) antibodies influences the response to abatacept, we compared therapeutic responses between anti-Ro/SSA antibody-negative and -positive patients with rheumatoid arthritis (RA) using a multicentre RA ultrasonography prospective cohort. Method: We reviewed Japanese patients with RA who started abatacept as the first biological disease-modifying anti-rheumatic drug between June 2013 and April 2018. We assessed 28-joint Disease Activity Score-erythrocyte sedimentation rate (DAS28-ESR) change between baseline and 6 or 12 months after treatment in RA patients treated with abatacept, and European League Against Rheumatism (EULAR) response at 6 and 12 months. The Global OMERACT-EULAR Synovitis Score (GLOESS) was calculated at baseline and at 6 and 12 months. Results: Overall, 51 patients were enrolled and divided into anti-Ro/SSA antibody-negative and -positive groups of 35 and 16, respectively. Median age at baseline was significantly higher in the anti-Ro/SSA antibody-negative group (p = 0.04). The retention rate and percentage of EULAR good responders at 12 months were significantly higher in the anti-Ro/SSA antibody-negative group (both p = 0.02). Anti-Ro/SSA antibody-negative patients exhibited larger decreases in both DAS28-ESR and DAS28-C-reactive protein at 12 months than anti-Ro/SSA antibody-positive patients (p = 0.02 and 0.04, respectively). GLOESS decreased significantly at 6 months in anti-Ro/SSA antibody-negative patients (p = 0.03). Multivariate analyses showed that anti-Ro/SSA antibody positivity was an independent factor associated with change in the DAS28-ESR at 6 months (p < 0.05). Conclusion: Anti-Ro/SSA antibody positivity predicts a poor response to abatacept and low retention rate.

Discrepancy between clinical and ultrasound remissions in rheumatoid arthritis: a multicentre ultrasound cohort study in Japan.

Objectives: Using multicentre ultrasound (US) cohort data among patients with rheumatoid arthritis (RA), we aimed to identify baseline factors that permit differentiation between two patient cohorts achieving US remission and clinical remission, and to determine the factors contributing to the discrepancy.Method: We reviewed 248 Japanese patients diagnosed with RA who underwent treatment with biological disease-modifying anti-rheumatic drugs at 13 centres. We performed US assessments of the synovia of 22 joints. We assessed the percentages of patients with clinical remission and US remission, defined as total power Doppler scores of 0 at 12 months.Results: The 87 patients who achieved US remission were divided into a group that achieved both clinical and US remission (n = 53) and a group that achieved US remission only (n = 34). Baseline factors that were significantly and independently associated with clinical remission at 12 months among patients who also achieved US remission included short disease duration, the presence of concomitant methotrexate use, and low patient global assessment score (p < 0.05, p < 0.05, and p < 0.005, respectively).Conclusions: RA patients with baseline high patient global assessment scores and long disease duration at baseline were unlikely to achieve clinical remission even after achieving US remission. Objective joint assessments using US provide additional information of potential importance for the management of RA.

Anti-citrullinated protein antibody titre as a predictor of abatacept treatment persistence in patients with rheumatoid arthritis: a prospective cohort study in Japan.

Objective: Successful rheumatoid arthritis (RA) outcome depends on treatment efficacy in the early stages of the disease and its sustainability. It is thus critical to identify factors predicting treatment persistence with biological agents, such as abatacept. We compared clinical profiles, including early changes in autoantibody titres at 3 months, between patients with RA demonstrating sustained persistence and those discontinuing abatacept treatment.Method: We prospectively enrolled 71 and 78 active RA patients treated with abatacept and tumour necrosis factor inhibitors (TNF-Is), respectively, who had previous disease-modifying anti-rheumatic drug) failure. Clinical characteristics were compared between non-continuation and continuation groups stratified according to abatacept or TNF-I persistence for at least 12 months from treatment initiation.Results: Significantly larger decreases in rheumatoid factor titre and anti-citrullinated protein autoantibody (ACPA) titre were observed in the continuation group of abatacept therapy at 3 months, and early reduction in ACPA titre remained a significant and independent predictor of sustained persistence with abatacept in multivariate analysis. In addition, we obtained the area under the receiver operator characteristics curve of 0.904 from a model including baseline ACPA titre and reduction of ACPA titre at 3 months. Sustained reduction of RA disease activity score at 12 months was significantly and independently associated with reduced ACPA titre at 3 months.Conclusions: Persistence with abatacept and sustained therapeutic response are associated with an early reduction in ACPA titre. Prediction of abatacept continuation and efficacy will facilitate the optimal design of therapy in the early stages of RA.

Changes in plasma concentrations of S100A7 and S100A8 in dairy cows during pregnancy.

This study was carried out to examine the changes in plasma concentrations of the Ca-binding antimicrobial proteins S100A7 and S100A8 during pregnancy in dairy cows. Holstein Friesian cows (n = 19) were inseminated with Holstein Friesian semen. Blood was collected at days 30, 60, 90, 120, 150, 180, 210, 240 and 270 after insemination. Plasma was used for measuring the concentrations of S100A7 and S100A8. Both S100A7 and S100A8 concentrations showed similar patterns during gestation; they increased during the midgestation, between days 90 and 180, and then declined before calving. The findings indicated that plasma concentrations of S100A7 and S100A8 did not change significantly during pregnancy in cows. Further studies are required to determine the roles of S100A7 and S100A8 in physiological function during pregnancy in dairy cows.

Possible mechanisms of the crosstalk between Langerhans cells and regulatory T cells in extramammary Paget disease by receptor activator of nuclear factor kappa B (RANK) ligand/RANK pathways.

BACKGROUND: Extramammary Paget disease (EMPD) is a skin adenocarcinoma of apocrine gland origin, in which Paget cells express receptor activator of nuclear factor kappa B (RANK) ligand (RANKL) and matrix metalloproteinase (MMP)-7, and release soluble (s)RANKL into the tumour microenvironment. We previously reported that about 60% of the RANK+ cells among the stromal cells are M2 macrophages, but the identity of the remaining population of RANK+ cells is still unknown. OBJECTIVES: To investigate the unknown subpopulation of RANK-expressing cells in EMPD. METHODS: The main population of RANK-expressing cells in the epidermis was composed of epidermal Langerhans cells (LCs). To explore the effects of RANKL on LCs, we stimulated LCs generated from human CD34+ hematopoietic progenitor cells with graded concentrations of sRANKL. To further examine the correlation between LCs and regulatory T cells (Tregs) in EMPD, we employed immunohistochemical staining. RESULTS: sRANKL stimulation was shown to augment the production of C-C motif chemokine ligand 17 (CCL17) from LCs. We additionally demonstrated CCL17 expression by CD1a+ LCs in EMPD in an immunofluorescence study. Spearman's rank correlation test confirmed a correlation between the number of LCs and the number of Foxp3+ Tregs in the lesional skin of invasive EMPD. In addition, the numbers of Foxp3+ Tregs in the sentinel lymph nodes of metastatic EMPD were significantly higher than those of metastatic melanoma, which did not express RANKL. CONCLUSIONS: The findings suggest that the RANKL/RANK pathway in EMPD might contribute to the recruitment of Tregs and to maintenance of the tumour microenvironment.

Removal of high concentration ammonia from wastewater by a combination of partial nitrification and anammox treatment.

Attached growth reactors were developed separately for solids retention time (SRT)-controlled partial nitrification and for anaerobic ammonia oxidation (Anammox) treatment, and a new nitrogen removal process is proposed for wastewater containing highly concentrated ammonia. For partial nitrification, an attached growth medium of polyurethane foam was used. Partial nitrification was achieved stably under a SRT of 4 days, and the abundance ratio of NO2(-)-N to the sum of NH4(+)-N, NO2(-)-N and NO3(-)-N was approximately 0.8 after 10 days. Under a SRT of4 days, the amoA gene concentrations of ammonia-oxidizing bacteria increased from 1 x 10(8) to 7 x 10(8) copies/l, whereas the 16S ribosomal RNA (16S rRNA) gene concentrations of nitrite-oxidizing bacteria did not increase. These results indicate that SRT-controlled operation is a promising technology for achieving partial nitrification. For the Anammox treatment, an attached growth medium of non-woven fabric was used. Inorganic nitrogen removal of approximately 80-90% was observed at an inorganic nitrogen loading rate of over 10 kgN/(m3-medium.d) and an influent nitrogen concentration of 400 mgN/l. Our non-woven fabric reactor showed similar or superior Anammox performance to that reported previously. By using a combination of these two rectors, we can develop a method that combines partial nitrification and Anammox treatment for effective and stable nitrogen removal.

Degradation characteristics of polylactide in thermophilic anaerobic digestion with hyperthermophilic solubilization condition.

To test whether hyperthermophilic treatment promotes polylactide (PLA) dissolution and methane conversion under anaerobic digestion conditions, a single thermophilic control reactor (55 °C) and a two-phase system consisting of a hyperthermophilic reactor (80 °C) and a thermophilic reactor (55 °C) were continuously fed with a mixture of PLA and artificial kitchen garbage. In Runs 1 and 2, the PLA dissolution ratios in the two-phase system were 79.2 ± 6.5% and 85.2 ± 7.0%, respectively, higher than those of the control. Batch experimental results indicated that hyperthermophilic treatment could promote PLA dissolution to a greater degree as compared with single thermophilic treatment and that ammonia addition also had a promotional effect on PLA dissolution. In the two-phase system, after hyperthermophilic treatment, dissolved PLA was converted to methane gas under the subsequent thermophilic condition.

Factors associated with possession of accurate knowledge regarding occupational health management among operations leaders of radiation decontamination workers in Fukushima, Japan: a cross-sectional study.

OBJECTIVES: An operations leader (OL) takes an important role in occupational health management for radiation decontamination workers in Japan, and candidates for the position must participate in a training session to acquire the necessary knowledge as required by law. However, it has not been clarified whether the candidates for the OL position actually possess accurate knowledge regarding occupational health management for such work after the training session. We, therefore, aimed at examining the current occupational health management knowledge among the candidates and investigating factors related to the knowledge, with hypothesis that possession of accurate knowledge is associated with prior experience of having worked in radiation decontamination. DESIGN: A cross-sectional study. SETTING: The training sessions held by Fukushima Prefecture Labor Standard Associations in Fukushima, Japan, in 2017. PARTICIPANTS: Eighty male candidates participated in the training sessions. OUTCOME: The number/proportion of correct answers to the questions regarding occupational health management, such as those on working environment management, control of operations and health management. RESULTS: The proportion of those who possessed accurate knowledge regarding working environment management, control of operations and health management was 68.8%, 55.0% and 51.2%, respectively. Experience of radiation decontamination work was associated with the possession of inaccurate knowledge regarding working environment management (OR 0.140 (95% CI 0.042 to 0.464)), and the uncertainty of future radiation decontamination work schedules in difficult-to-return zones was associated with the possession of accurate knowledge regarding health management (OR 4.344 (95% CI 1.509 to 12.50)). CONCLUSIONS: Previous experience in radiation decontamination work may hinder the ability to acquire accurate information regarding working environment management among candidates for an OL position. To promote adequate occupational health management for radiation decontamination workers, it is required to establish an effective instructional method for the OL candidate training sessions with consideration of previous relevant experience.

Genotoxicity studies on licorice flavonoid oil (LFO).

Licorice flavonoid oil (LFO) is a new functional food ingredient. In this study, the genotoxicity of LFO was investigated using a test battery of three different methods. In a reverse mutation assay using four Salmonella typhimurium strains and Escherichia coli, LFO did not increase the number of revertant colonies in any tester strain with or without metabolic activation by rat liver S9 mix. In a chromosomal aberration test using Chinese hamster lung (CHL/IU) cells, LFO did not induce any chromosomal aberrations either in the short period test without rat liver S9 mix or in the continuous treatment (24 h or 48 h) test. However, in the short-period test with rat liver S9 mix, LFO induced structural chromosomal aberrations at concentrations higher than 0.6 mg/mL. A bone marrow micronucleus test using male F344 rats was initially conducted. The animals were dosed by oral gavage at doses up to 5000 mg/kg/day. No significant or dose-dependent increases in the frequency of micronucleated polychromatic erythrocytes (MNPCE) were observed and the high dose suppressed the ratio of polychromatic erythrocytes (PCE) to total erythrocytes. Subsequently, a liver and peripheral blood micronucleus test using male F344 rats was conducted. No micronuclei induction either in hepatocytes or PCE was observed even at the highest dose of 5000 mg/kg/day. From the findings obtained from the genotoxicity assays performed in this study and the published pharmacokinetic studies of LFO, it appears unlikely that dietary consumption of LFO will present any genotoxic hazard to humans.

90-Day repeated-dose toxicity study of licorice flavonoid oil (LFO) in rats.

Licorice flavonoid oil (LFO) is a new functional food ingredient consisting of licorice hydrophobic polyphenols in medium-chain triglycerides (MCT). As part of a safety evaluation, a 90-day oral toxicity study in rats was conducted using an LFO concentrate solution (2.90% glabridin). Male and female animals were assigned to one of 12 groups (10 males or females per group) and received corn oil (negative control), MCT (vehicle control), or 400, 600, 800 or 1600 mg/kg of the LFO concentrate solution. In conclusion, LFO concentrate solution induced an anticoagulation effect in both sexes, although there was a clear sex difference. Based on these findings, it is concluded that the no-observed-adverse-effect level (NOAEL) for the LFO concentrate solution is estimated to be 800 mg/kg/day for female rats, and approximately 400 mg/kg/day for male rats.

The performance and microbial diversity of temperature-phased hyperthermophilic and thermophilic anaerobic digestion system fed with organic waste.

The objective of this study was to evaluate the performances and microbial diversities for development of the effective hyperthermophilic digester system that consists of a hyperthermophilic reactor and hyperthermophilic or thermophilic reactor in series. Lab-scale reactors were operated continuously fed with artificial kitchen garbage. The effect of temperature on the acidification step was firstly investigated. Results indicated that 20.8% of COD solubilization was achieved at 70 degrees C, with 12.6% at 80 degrees C. The average protein solubilization reached 31% at 80 degrees C. Methane conversion efficiency following the acidification was around 85% on average at 55 degrees C, but decreased with increasing temperature and methane gas was not produced over 73 degrees C. As well, bacteria affiliated with the methanogens dominated the population below 65 degrees C, while those affiliated with acidogens were predominant over 73 degrees C. These results indicated that the hyperthermophilic process has considerable benefits to treat wastewater or waste containing high concentration of protein.

Omission of lymphadenectomy is possible for low-risk corpus cancer.

AIM: The objective of this study is to ascertain whether omission of lymphadenectomy is possible when endometrial cancer is considered low-risk based on intraoperative pathologic indicators. PATIENT AND METHODS: A total of 128 patients were deemed to be low-risk based on intraoperative evaluation, including frozen-section determination of grade and myometrial invasion. We divided these 128 patients into 2 groups, the total abdominal hysterectomy and bilateral salpingo-oophorectomy (TAH-BSO) with lymphadenectomy (LA group, n=68) and the TAH-BSO without lymphadenectomy (non-LA group, n=60) group. The only adjuvant treatment used was chemotherapy, and the decision to use chemotherapy was based on stage, grade, or lymphovascular space involvement. A retrospective review of the medical records was performed, and disease-free survival (DFS), overall survival (OS), operative time, estimated blood loss during operation, percentage of transfusion requirement, incidence of post-operative leg lymphedema and post-operative deep vein thrombosis were evaluated. RESULTS: The 5-year DFS and OS rates were 95.6% and 98.5% in the LA group, and 98.3% and 98.3% in the non-LA group, respectively, and were not significantly different. In the LA group, pelvic lymph node metastasis was observed in only 1 case. In the LA group, blood loss during operation, percentage of transfusion requirement and the incidence of post-operative leg lymphedema were significantly higher than those in the non-LA group. CONCLUSION: Lymphadenectomy did not provide a significant survival advantage in the patients with low-risk corpus cancer. Additionally, some peri- and post-operative morbidities and complications were increased by the addition of lymphadenectomy. The present findings suggest that lymphadenectomy should be limited for low-risk corpus cancer.

A multicenter study of leukocytapheresis in rheumatoid arthritis.

OBJECTIVE: To evaluate the efficacy and safety of leukocytapheresis (LCAP) in patients with rheumatoid arthritis (RA) that is refractory to disease modifying antirheumatic drugs (DMARDs), we conducted a prospective, multicenter, open-label clinical trial. METHODS: We enrolled 38 active RA patients, including 32 patients who showed an inadequate response to > or = 2 DMARDs and 6 patients with rapidly progressive RA. All patients continued drug therapy and were treated with 5 LCAP sessions conducted at 1-week intervals. The clinical response was evaluated at baseline before starting LCAP and at 4 weeks after the completion of all the LCAP sessions using the American College of Rheumatology (ACR) criteria and the 28-joint disease activity score (DAS28) of the European League Against Rheumatism (EULAR). RESULTS: Of the 35 patients who fulfilled the study's eligibility criteria, 24 (69%), 10 (29%), and 23 (66%) patients achieved 20% (ACR20), 50% (ACR50), and DAS28-C-reactive protein (CRP) EULAR improvement, respectively. The mean DAS28-CRP score of the 35 patients decreased significantly from 5.99 +/- 0.92 at baseline to 4.54 +/- 1.39 after treatment. Comparison analysis of the ACR20 responders and non-responders to LCAP revealed that 22 of 24 responders (92%) concomitantly received methotrexate, whereas significantly fewer, that is, 6 of 11 non-responders (55%) received methotrexate. Less frequent and transient mild-to-moderate adverse events, including nausea and headache, were seen in 12 of 189 LCAP sessions (6.3%). CONCLUSION: These results demonstrate the usefulness of LCAP in combination with DMARDs, particularly methotrexate, as an effective and safe treatment for refractory RA.

Microbial population dynamics in a thermophilic methane digester fed with garbage.

The diversity of microbial communities in three full-scale thermophilic anaerobic digesters which treated garbage, sewage sludge and livestock wastes (hereafter called TGD, TSD and TLD, respectively) was investigated using 16S rDNA clone libraries in triplicate. The population dynamics of TGD were also studied. The purposes were to show the microbial diversity in each reactor and to suggest which key microbes in a thermophilic methane digester fed with garbage, including a check of reproducibility and the suggestion of an error range in this molecular biology method. 736 clones were identified, and the maximum error was estimated to be around +/-10% for the same OTU (operational taxonomic unit) and for most detected OTUs. The most frequently detected OTU shows a close relationship to Uncultured bacterium clone MBA08, Unidentified bacterium clone TUG22 and Uncultured archaeal symbiont PA204 in TGD, TSD and TLD, respectively. The microbial population dynamics in TGD were studied over a period of 90 days, and the occupying ratios of Bacillus infernus and Methanothermobacter wolfeii were shown to change with the change in VFA concentration. From the dynamic change and characteristics of the microbes, it is concluded that Bacillus infernus and Methanothermobacter wolfeii played an important role and were recommended as key microbes in TGD.

Calreticulin mutant mice develop essential thrombocythemia that is ameliorated by the JAK inhibitor ruxolitinib.

Mutations of calreticulin (CALR) are detected in 25-30% of patients with essential thrombocythemia (ET) or primary myelofibrosis and cause frameshifts that result in proteins with a novel C-terminal. We demonstrate that CALR mutations activated signal transducer and activator of transcription 5 (STAT5) in 293T cells in the presence of thrombopoietin receptor (MPL). Human megakaryocytic CMK11-5 cells and erythroleukemic F-36P-MPL cells with knocked-in CALR mutations showed increased growth and acquisition of cytokine-independent growth, respectively, accompanied by STAT5 phosphorylation. Transgenic mice expressing a human CALR mutation with a 52 bp deletion (CALRdel52-transgenic mice (TG)) developed ET, with an increase in platelet count, but not hemoglobin level or white blood cell count, in association with an increase in bone marrow (BM) mature megakaryocytes. CALRdel52 BM cells did not drive away wild-type (WT) BM cells in in vivo competitive serial transplantation assays, suggesting that the self-renewal capacity of CALRdel52 hematopoietic stem cells (HSCs) was comparable to that of WT HSCs. Therapy with the Janus kinase (JAK) inhibitor ruxolitinib ameliorated the thrombocytosis in TG mice and attenuated the increase in number of BM megakaryocytes and HSCs. Taken together, our study provides a model showing that the C-terminal of mutant CALR activated JAK-STAT signaling specifically downstream of MPL and may have a central role in CALR-induced myeloproliferative neoplasms.

Evaluation of the mutagenic potential of ubidecarenone using three short-term assays.

Addition of ubidecarenone, coenzyme Q(10) (CoQ(10)), to foods has been proposed for its nutritive value. Ubidecarenone is present naturally in a number of foods, including meats (e.g., beef, chicken) and fish (e.g., herring, rainbow trout), and on average, people are estimated to consume 2-20 mg/day of this metabolically important substance. Currently, relatively little formal evidence regarding the safety of ubidecarenone has been identified in the toxicology literature, despite its consumption by humans for centuries without reported notable adverse effects. As such, a series of toxicological studies, including mouse bone marrow micronucleus, chromosomal aberration, and bacterial reverse mutation tests, were conducted to evaluate the in vivo and in vitro mutagenic potential of CoQ(10). The test article, ubidecarenone, was devoid of clastogenic activity when administered orally to mice at doses up to 2000 mg/kg/day. In addition, the test article did not induce chromosomal aberration in CHL/IU cells exposed to concentrations as great as 5.0 mg/ml, nor did it induce reverse mutations in Salmonella typhimurium and Escherichia coli at concentrations as great as 5000 microg/plate.

Electron spin resonance studies on the mechanism of adriamycin-induced heart mitochondrial damages.

Rats were given i.p. injections of Adriamycin (4 mg/kg body weight) for 6 consecutive days. Electron spin resonance spectrometry with spin labeling and a trapping technique was applied to heart mitochondria obtained from treated and control animals, in order to examine the physical response of heart mitochondrial membrane affected by Adriamycin. The Adriamycin treatment resulted in a decrease of membrane fluidity (and an increase in order parameter S), with concomitant dysfunction of respiratory responses. The generation of hydroxyl radicals from mitochondria was enhanced in the Adriamycin-treated group. In addition, superoxide dismutase activity in the mitochondrial matrix was found to decrease. The heart mitochondria of Adriamycin-treated animals contained a large amount of lipid peroxide. These results suggest that the enhancement of hydroxyl radical formation in mitochondria affected by Adriamycin is one of the factors involved in Adriamycin-induced cardiotoxicity.

Stimulation of polyadenosine diphosphoribose synthesis by DNA lesions induced by sodium chromate in Chinese hamster V-79 cells.

Polyadenosine diphosphoribose [poly(ADP-ribose)] synthesis was stimulated by DNA lesions induced with Na2CrO4 and methyl methanesulfonate (MMS) in Chinese hamster V-79 cells. Na2CrO4 and MMS induced DNA single-strand breaks in a concentration-dependent manner; however, the breaks induced by Na2CrO4 were "protein associated" while those induced by MMS were not. MMS stimulated in a dose-dependent fashion the synthesis of poly(ADP-ribose) up to 6-fold above the control. Na2CrO4 also induced poly(ADP-ribose) synthesis, but the level of synthesis was less than 3-fold. Control experiments demonstrated that Na2CrO4 treatment of cells did not affect their ability to synthesize poly(ADP-ribose) in response to DNA damage. Treatment of cells with Na2CrO4 and MMS induced more poly(ADP-ribose) synthesis than each agent alone; however, whenever Na2CrO4 was utilized, the breaks required proteinase K to be detected. Following removal of extracellular chromate, the DNA strand breaks induced by 0.2 mM Na2CrO4 were repaired quickly during the first hour but more slowly for the next 3 h. In the presence of 3-aminobenzamide, an inhibitor of poly(ADP-ribose) synthesis, the repair of DNA breaks was reduced. These results suggest that DNA protein-associated breaks produced by Na2CrO4 were recognized by poly(ADP-ribose) polymerase and that there are differences in poly(ADP-ribose) synthesis in response to Na2CrO4 and MMS. The results also suggest that the repair of breaks induced by Na2CrO4 are associated with poly(ADP-ribose) synthesis, but perhaps because most of these breaks are protein associated, there is less stimulation of poly(ADP-ribose) synthesis.