Detailed Dose-response Analyses Research Articles

Rosiglitazone Does Not Affect the Risk of Inflammatory Bowel Disease: A Retrospective Cohort Study in Taiwanese Type 2 Diabetes Patients.

Human studies on the effect of rosiglitazone on inflammatory bowel disease (IBD) are still lacking. We investigated whether rosiglitazone might affect IBD risk by using the reimbursement database of Taiwan's National Health Insurance to enroll a propensity-score-matched cohort of ever users and never users of rosiglitazone. The patients should have been newly diagnosed with diabetes mellitus between 1999 and 2006 and should have been alive on 1 January 2007. We then started to follow the patients from 1 January 2007 until 31 December 2011 for a new diagnosis of IBD. Propensity-score-weighted hazard ratios were estimated with regards to rosiglitazone exposure in terms of ever users versus never users and in terms of cumulative duration and cumulative dose of rosiglitazone therapy for dose-response analyses. The joint effects and interactions between rosiglitazone and risk factors of psoriasis/arthropathies, dorsopathies, and chronic obstructive pulmonary disease/tobacco abuse and the use of metformin were estimated by Cox regression after adjustment for all covariates. A total of 6226 ever users and 6226 never users were identified and the respective numbers of incident IBD were 95 and 111. When we compared the risk of IBD in ever users to that of the never users, the estimated hazard ratio (0.870, 95% confidence interval: 0.661-1.144) was not statistically significant. When cumulative duration and cumulative dose of rosiglitazone therapy were categorized by tertiles and hazard ratios were estimated by comparing the tertiles of rosiglitazone exposure to the never users, none of the hazard ratios reached statistical significance. In secondary analyses, rosiglitazone has a null association with Crohn's disease, but a potential benefit on ulcerative colitis (UC) could not be excluded. However, because of the low incidence of UC, we were not able to perform detailed dose-response analyses for UC. In the joint effect analyses, only the subgroup of psoriasis/arthropathies (-)/rosiglitazone (-) showed a significantly lower risk in comparison to the subgroup of psoriasis/arthropathies (+)/rosiglitazone (-). No interactions between rosiglitazone and the major risk factors or metformin use were observed. We concluded that rosiglitazone has a null effect on the risk of IBD, but the potential benefit on UC awaits further investigation.

Novel Insights into Pyrrolizidine Alkaloid Toxicity and Implications for Risk Assessment: Occurrence, Genotoxicity, Toxicokinetics, Risk Assessment-A Workshop Report.

This paper reports on the major contributions and results of the 2nd International Workshop of Pyrrolizidine Alkaloids held in September 2020 in Kaiserslautern, Germany. Pyrrolizidine alkaloids are among the most relevant plant toxins contaminating food, feed, and medicinal products of plant origin. Hundreds of PA congeners with widespread occurrence are known, and thousands of plants are assumed to contain PAs. Due to certain PAs' pronounced liver toxicity and carcinogenicity, their occurrence in food, feed, and phytomedicines has raised serious human health concerns. This is particularly true for herbal teas, certain food supplements, honey, and certain phytomedicinal drugs. Due to the limited availability of animal data, broader use of in vitro data appears warranted to improve the risk assessment of a large number of relevant, 1,2-unsaturated PAs. This is true, for example, for the derivation of both toxicokinetic and toxicodynamic data. These efforts aim to understand better the modes of action, uptake, metabolism, elimination, toxicity, and genotoxicity of PAs to enable a detailed dose-response analysis and ultimately quantify differing toxic potencies between relevant PAs. Accordingly, risk-limiting measures comprising production, marketing, and regulation of food, feed, and medicinal products are discussed.

Erythrocyte Cytochrome b Reductase 1 Protein Levels and Structure in Sickle Cell Patients

In humans, vitamin C (ascorbic acid) is required as substrate or cofactor in a number of important cellular processes. Patients with sickle cell disease (SCD) often are deficient in vitamin C and thus potentially adversely affected. A major determinant of the cellular availability of ascorbate is the plasma level of the vitamin. This reflects the balance between dietary supply, uptake by tissues, excretion, oxidative reactions in the plasma, and RBC dependent recycling or irreversible decomposition of oxidized ascorbate. The mechanisms for depressed plasma ascorbate levels in SCD patients have not been identified, and the effectiveness and safety of dietary ascorbate supplements have not been established. As RBC dysfunction is central to SCD, we have focused on the possibility that SCD patients' RBC membranes have an altered level, structure or function of CYBRD1, a key enzyme in recycling oxidized plasma ascorbate (Fig. 1A).Materials and methods:Blood samples were collected in EDTA tubes from adult SCD patients at routine ambulatory clinic visits and from healthy African American adults. Protocols were approved by institutional review. RBC were isolated by centrifugation and lysed in hypotonic buffer; membranes were isolated by centrifugation and stored at -80 C.Electrophoretic and western blot analyses: RBC membrane proteins were separated by SDS-PAGE, stained with Coomassie Blue and analyzed by densitometry to quantitate total membrane protein (standard: BSA). Alternatively, separated proteins were transferred to nitrocellulose or PVDF and probed with antibodies against CYBRD1. Immunoreactive bands were visualized colorimetrically and quantitated by densitometry (standard: recombinant human CYBRD1 (rCYBRD1)).Mass spectroscopic analyses: CYBRD1 was extracted from RBC membranes with dodecyl maltoside, and further enriched by immunoprecipitation. The immunopurified mixture was separated by SDS-PAGE and stained with Coomassie Blue. Gel pieces containing the CYBRD1 monomers were subjected to in-gel digestion and MS analysis of the tryptic peptides.Results and conclusions:Detailed dose-response analyses found unsuspected positively cooperative behavior between 0 and ~2 ng CYBRD1 in both rCYBRD1 and RBC membrane samples. This invalidated calculations of RBC CYBRD1 content based on linear response to rCYBRD1. However, selected RBC membrane samples were routinely included as quality controls on many blots. Further, the dose-response curves for RBC membrane samples had a consistent shape that fit a simple saturable model with an x-axis offset parameter. It was thus possible to estimate the CYBRD1 content for 36 of the RBC samples using one of them (V7) as reference (Fig. 1B). This permitted our ongoing comparison of CYBRD1 content in patients and controls, and in SCD subtypes.RBC CYBRD1 content in 4 homozygous SCD subjects sampled at 5 regular clinic visits changed little over 7-9 months (averages: 0.32 ± 0.05; 0.25 ± 0.05; 0.41 ± 0.04; and 0.37 ± 0.04 ng CYBRD1/µg RBC membrane protein). This suggests that patients' RBC CYBRD1 contents are relatively stable despite the short RBC half-life, and that a single sampling is sufficient for cross sectional studies.There was no significant difference in CYBRD1 level among ambulatory HbSC (N=6) and HbSS (N=8) patientsand healthy controls (N=8)(Fig. 1C, top). However, there were notable differences in CYBRD1 protein modification, with significantly less modification in CYBRD1 from HbSS patients than either HbSC patients or healthy controls (Fig. 1C, bottom).Similar isoform banding patterns are seen for patient and control CYBRD1 with antibodies against either the N- and C-terminal peptides (Fig. 1D). Retention of epitopes at both ends of CYBRD1 in the isoforms argues against differential proteolysis being involved. The mass spectrometric analyses found extensive oxidative modification of multiple methionine residues, but similar patterns were seen in RBC CYBRD1 from an SCD patient and a healthy control. Phosphorylation was found at multiple sites in residues 200-285 of CYBRD1 from both patient and control, but the fraction phosphorylated appeared much too low to account for the large proportion of CYBRD1 in individual isoforms. Thus, the structural basis for the isoforms' differential gel mobility remains to be identified.Support: American Heart Association, 16GRNT29170013 (R. Kulmacz); NIH 5T35DK007676 (B. Kone) [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mode-of-action evaluation for the effect of trans fatty acids on low-density lipoprotein cholesterol

The purpose of this work is to systematically consider the data relating to the mode of action (MOA) for the effects of industrially produced trans fatty acid (iTFA) on plasma low-density lipoprotein (LDL) levels. The hypothesized MOA is composed of two key events: increased LDL production and decreased LDL clearance. A substantial database supports this MOA, although the key events are likely to be interdependent, rather than sequential. Both key events are functions of nonlinear biological processes including rate-limited clearance, receptor-mediated transcription, and both positive and negative feedback regulation. Each key event was evaluated based on weight-of-evidence analysis and for human relevance. We conclude that the data are inadequate for a detailed dose-response analysis in the context of the evolved Bradford Hill considerations; however, the weight of evidence is strong and the overall shape of the dose-response curves for markers of the key events and the key determinants of those relationships is well understood in many cases and is nonlinear. Feedback controls are responsible for maintaining homeostasis of cholesterol and triglyceride levels and underlie both of the key events, resulting in a less-than-linear or thresholded relationship between TFA and LDL-C. The inconsistencies and gaps in the database are discussed.

Metabolically Healthy Obesity and the Development of Nonalcoholic Fatty Liver Disease.

The risk of nonalcoholic fatty liver disease (NAFLD) among obese individuals without obesity-related metabolic abnormalities, a condition referred to as metabolically healthy obese (MHO), is largely unexplored. Therefore, we examined the association between body mass index (BMI) categories and the development of NAFLD in a large cohort of metabolically healthy men and women. A cohort study was conducted in 77,425 men and women free of NAFLD and metabolic abnormalities at baseline, who were followed-up annually or biennially for an average of 4.5 years. Being metabolically healthy was defined as not having any metabolic syndrome component and having a homeostasis model assessment of insulin resistance <2.5. The presence of fatty liver was determined using ultrasound. During 348,193.5 person-years of follow-up, 10,340 participants developed NAFLD (incidence rate, 29.7 per 1,000 person-years). The multivariable adjusted hazard ratios (95% confidence intervals) for incident NAFLD comparing overweight and obese with normal-weight participants were 2.15 (2.06-2.26) and 3.55 (3.37-3.74), respectively. In detailed dose-response analyses, increasing baseline BMI showed a strong and approximately linear relationship with the incidence of NAFLD, with no threshold at no risk. This association was present in both men and women, although it was stronger in women (P for interaction <0.001), and it was evident in all clinically relevant subgroups evaluated, including participants with low inflammation status. In a large cohort of strictly defined metabolically healthy men and women, overweight and obesity were strongly and progressively associated with an increased incidence of NAFLD, suggesting that the obese phenotype per se, regardless of metabolic abnormalities, can increase the risk of NAFLD.

Defining Primary Marrow Microenvironment-Induced Synthetic Lethality and Resistance for 2,684 Approved Drugs Across Molecularly Distinct Forms of Multiple Myeloma

▪Multiple Myeloma (MM) is a prototypical neoplasm for the study of tumor-microenvironment interactions and influences on drug response. These interactions within the bone marrow (BM) alter the signaling state of MM cells and their relative dependence on pharmacological targets. Conversely, many efforts to identify and validate drug targets in MM are conducted outside of this context. This raises the possibility that systematic re-evaluation of the current pharmacopeia may identify drugs with previously unappreciated capacity for targeting MM cells within the marrow environment.To this end, we utilized the compartment-specific bioluminescence platform (CS-BLI) to characterize the activity of 2,684 FDA-approved drugs from The Johns Hopkins Drug Library (JHDL) in three distinct MM subtypes, in the presence or absence of patient-derived CD138-negative bone marrow stromal cells (BMSCs). Anti-MM activity was evaluated at 100 nM concentrations over 72 h in MM1S (t(14;16), KRASG12A, TRAF3LOF), L363 (t(20;22), NRASQ61H, p53S261T), and OPM2 (t(4;14), FGFRK560E, p53R175H) lines. These lines demonstrate phenotypes of strong, medium, and low BMSC-induced growth enhancement, respectively. Active drugs were placed into 4 categories: type 1 - having potent anti-MM activity independent of BMSC interactions (no stromal effect), type 2 - having anti-MM activity only in the presence of BMSCs (stroma-dependent "synthetic lethality"), type 3 - having anti-MM activity that is decreased in the context of BMSCs (stroma-dependent resistance), and type 4 - otherwise inactive agents that demonstrate pro-MM activity in context of BMSCs (stroma-dependent stimulants).In this study, for MM1S, L363, and OPM2, respectively, we identified 103, 118, and 108 type 1 drugs, 217, 105, and 76 type 2 drugs, 128, 75, and 16 type 3 drugs, and 124, 33, and 38 type 4 drugs. For each category of drug phenotype, we assessed overlap across the three MM cell lines. We observed high degree of overlap for type 1 drugs (67 drugs active in all three models), while more diversity between lines was evident across the 3 lines for type 2-4 drugs, whose activity is altered by interaction with BMSCs (Figure 1). Specifically, focusing on agents demonstrating BMSC-associated stimulation, adrenergic drugs consistently stimulated MM growth in context of BMSCs, while glucocorticoids consistently grouped as type 3 agents (demonstrating BMSC-associated resistance). Interestingly, carfilzomib was also subject to BMSC-associated resistance.Despite differences in drugs demonstrating stroma-induced lethality across the MM cell lines, salicylates were commonly represented in this category. In addition to the salicylates, tofacitinib, a Janus kinase (JAK) inhibitor, demonstrated a strong capacity to elicit a stroma-dependent synthetic lethal phenotype and ruxolitinib, another inhibitor in the same class, showed a similar, yet distinct pattern of stroma-mediated sensitization.In conjunction with our screen, we performed an RNA-seq analysis to assess differential gene expression between MM in monoculture vs. in co-culture with BMSCs. Expression analysis revealed 4.0 fold increase in JAK3 expression induced by co-culture with primary BMSCs, as well as induction of a STAT3 transcription factor fingerprint by ChIP-seq enrichment analysis. A detailed dose-response analysis of tofacitinib revealed no anti-MM activity against MM cells in isolation at physiological concentrations, but showed typical sigmoidal log-dose response dynamics in the presence of stroma and a dynamic range that completely abrogated the growth advantage attributable to stromal stimulation.This phenomenon of BMSC-dependent pharmacology identifies tofacitinib as an intriguing candidate for repurposing as an agent demonstrating stroma-induced synthetic lethality against MM. Further evaluation of this agent in combination with other anti-MM agents, like bortezomib, is also warranted. Taken together, this study demonstrates specific anti-MM activity for a wide array of clinically relevant drugs and drug classes in the context of BM microenvironment interactions and provides context for further validation and potential suitability for repurposing to treat MM within the medullary compartment. [Display omitted] DisclosuresAftab:Cleave Biosciences, Inc.: Research Funding; Omniox, Inc.: Research Funding; Atara Biotherapeutics, Inc.: Employment, Equity Ownership; Onyx Pharmaceuticals, Inc.: Research Funding. Off Label Use: The use of tofacitinib citrate and ruxolitinib will be discussed in preclinical contexts for treatment of multiple myeloma. Other approved drugs and drug classes will be generally presented in similar off-label context..

Calcium intake and bone mineral density as an example of non-linearity and threshold analysis.

Non-linearity is a likely phenomenon in bone metabolism, but is often ignored in pertinent epidemiological studies. Using NHANES III data on calcium intake and bone mineral density, the most important non-linear methods are introduced and discussed. The results should motivate researchers to consider non-linearity in this field more frequently. Many relationships in bone metabolism and homeostasis are likely to follow non-linear patterns. Detailed dose-response analyses allowing for non-linear associations nonetheless remain scarce in this field. A detailed analysis of NHANES III data on dietary calcium intake and bone mineral density was used to demonstrate the application and some of the challenges of the most important dose-response methods, including LOESS, categorical analysis, fractional polynomials, restricted cubic splines, and segmented regression. The spline estimate suggested increasing bone mineral density up to a calcium intake of about 1 g/day and a plateau thereafter. In segmented regression, the break-point marking the beginning of the plateau was placed at an intake of 0.58 (95 % confidence interval, 0.33 to 0.82) g/day. Sensitivity analyses suggested a less curved dose-response in women. Knowing about the possibilities and limitations of non-linear dose-response approaches should encourage researchers to consider these methods more frequently in studies on bone health and disease. The example analysis suggested bone mineral density to reach a plateau slightly below current calcium intake recommendations, with fairly pronounced differences of the dose-response shape by sex and menopausal status.

Inheritance of resistance to 2,4-D and chlorsulfuron in a multiple-resistant population of Sisymbrium orientale.

A population of Sisymbrium orientale from South Australia has multiple resistance to auxinic herbicides and inhibitors of acetohydroxyacid synthase (AHAS). Inheritance of resistance to 2,4-D and chlorsulfuron was studied in this population. Crosses were made between seven resistant individuals as pollen donors to seven susceptible individuals. Sixteen F1 individuals from three crosses were identified by their lack of strong epinasty when treated with 200 g 2,4-D ha(-1). These individuals were selfed, and segregation analysis of strong epinasty in the resulting progeny fitted a 3:1 ratio for resistant:susceptible individuals when treated with 200 g 2,4-D ha(-1), as predicted by a single major gene. A detailed dose-response analysis of the F2 populations to 2,4-D confirmed single-gene inheritance. Analysis of segregation to 1 g chlorsulfuron ha(-1), a concentration that kills all susceptible individuals, was unable to determine the mode of inheritance. A detailed dose-response analysis indicated that two genes contributed to chlorsulfuron resistance: a dominant target-site mutation of Pro 197 to Ser and a second gene with dose-dependent dominance. This population has a single dominant allele conferring 2,4-D resistance, whereas two genes contribute to chlorsulfuron resistance. Single dominant gene inheritance demonstrates that 2,4-D resistance can be readily selected.

Smoking as an effect modifier of the association of calcium intake with bone mineral density.

Data from physiological studies suggest smoking to have detrimental effects on calcium absorption, but large-scale investigations of this interaction and its importance with respect to bone health in humans are lacking. The objective of the study was to examine the potential smoking-associated effect heterogeneity in the relationship of dietary calcium intake with bone mineral density in the general population. This was an observational, cross-sectional study. A total of 14 116 participants of the Third National Health and Nutrition Examination Survey, including 6882 never, 3532 former, and 3702 current smokers. The median age of the participants was 44 years, and 52% were female. Bone mineral density at the femoral neck as determined by dual-energy x-ray absorptiometry was measured. In multiple linear regression adjusting for age, sex, race/ethnicity, and education, the association of calcium intake with bone mineral density was characterized by suggestive overall positive trends in all three smoking behavior categories. Detailed dose-response analyses by restricted cubic spline modeling revealed more pronounced nonlinearity among former smokers, but the interaction of smoking with calcium intake on bone mineral density did not reach statistical significance in any of these models. The dose-response curves became even more homogenous across smoking behavior strata after additional adjustment for body mass index and physical activity. Even though the present results cannot rule out that smoking-associated differences in calcium absorption exist, they do suggest that smoking behavior does not have any relevant impact on the beneficial effects of calcium intake on bone mineral density at the population level.

Serum parathyroid hormone and risk of adverse outcomes in patients with stable coronary heart disease

Background and objectiveRecent longitudinal studies have suggested an association of high serum parathyroid hormone levels (PTH) with elevated cardiovascular risk in the general population. This study presents analyses of the...

REPROCESSED URANIUM EXPOSURE AND LUNG CANCER RISK

This study investigated the risk of lung cancer in regards to protracted occupational exposure to reprocessed uranium compounds. Two thousand seven hundred and nine male workers employed at the AREVA NC uranium processing plant between 1960 and 2005 in France were included in the cohort. Historical exposure to reprocessed uranium compounds classified by their solubility type was assessed on the basis of the plant's specific job-exposure matrix. Cox proportional hazard models adjusted for attained age, calendar period, and socioeconomic status were used to estimate relative risks in regards of each type of uranium compound. The relative risk of lung cancer tended to increase with decreasing solubility of reprocessed uranium compounds. The highest-though not statistically significant-relative risk was observed among workers exposed to slowly soluble reprocessed uranium dioxide. This study is the first suggesting an increasing risk of lung cancer associated with exposure to reprocessed uranium. Our results are consistent with data from experimental studies of biokinetics and the action mechanism of slowly soluble uranium compounds, but need to be confirmed in larger studies with more detailed dose-response analyses.

Evaluation of protein allergenic potential in mice: dose-response analyses.

With the increasing interest in novel foods derived from transgenic crop plants, there is a growing need for the development of approaches for the characterization of the allergenic potential of proteins. Whereas immunogenicity is a common property of foreign proteins, including food proteins, relatively few are significant dietary allergens with the inherent capacity to provoke IgE antibody production and immediate-type hypersensitivity responses. In order to evaluate an approach for the measurement of the allergenic potential of proteins, detailed dose-response analyses of humoral immune responses induced following systemic exposure of BALB/c strain mice to proteins known to differ in terms of sensitizing activity have been conducted. Mice were exposed to a range of concentrations of ovalbumin, a major allergenic constituent of hen's egg, a purified peanut allergen, Arachis hypogea agglutinin, or to the milk allergen bovine serum albumin, and to materials considered to lack significant allergenicity: a crude potato protein extract and a purified potato protein, potato agglutinin. The specific IgE antibody was measured by homologous passive cutaneous anaphylaxis assay, and the specific IgG antibody was measured by enzyme-linked immunosorbent assay. Each of the five proteins was immunogenic in mice, inducing IgG antibody responses at all doses tested, although there was some variation with respect to the vigour of IgG responses. Marked differences in the capacity of these proteins to induce IgE responses were observed, however, with relatively high-titre IgE antibody provoked by all three allergens over the dose ranges examined, whereas the potato proteins stimulated low-titre IgE antibody at the highest dose (10%) only. Importantly, differences in IgE antibody production have been observed against a background of equivalent immunogenicity (IgG antibody responses). The data presented here suggest that the measurement of antibody (IgE) responses in BALB/c mice appears to identify allergens accurately and to distinguish them from those materials that apparently lack allergenicity.

Collection, Validation, and Treatment of Data for a Mortality Study of Nuclear Industry Workers

Abstract To investigate the long-term health effects of protracted occupational exposure to low levels of ionizing radiation, a combined facility mortality study was initiated for 118,588 workers hired between 1942 and 1982 by three Department of Energy facilities in Oak Ridge, Tennessee. The primary objectives of the study were: (1) to evaluate and compare the mortality experience of separate subcohorts delineated by facility of employment, and (2) to conduct detailed dose-response analyses of the combined facilities subcohort having potential for external radiation exposure. Presented here are issues involving validation and treatment of data for study members, and characteristics of their radiation exposure. To verify data accuracy a stratified random sample was chosen, and original source documents containing demographic and radiation exposure data were reviewed. Health physicists investigated monitoring policies and practices in place at each facility over the 42 years of follow-up (1943 to 1984) bef...