Hubert Dirven

A primer on systematic reviews in toxicology

Systematic reviews, pioneered in the clinical field, provide a transparent, methodologically rigorous and reproducible means of summarizing the available evidence on a precisely framed research question. Having matured to a well-established approach in many research fields, systematic reviews are receiving increasing attention as a potential tool for answering toxicological questions. In the larger framework of evidence-based toxicology, the advantages and obstacles of, as well as the approaches for, adapting and adopting systematic reviews to toxicology are still being explored. To provide the toxicology community with a starting point for conducting or understanding systematic reviews, we herein summarized available guidance documents from various fields of application. We have elaborated on the systematic review process by breaking it down into ten steps, starting with planning the project, framing the question, and writing and publishing the protocol, and concluding with interpretation and reporting. In addition, we have identified the specific methodological challenges of toxicological questions and have summarized how these can be addressed. Ultimately, this primer is intended to stimulate scientific discussions of the identified issues to fuel the development of toxicology-specific methodology and to encourage the application of systematic review methodology to toxicological issues.

Exposure to perfluoroundecanoic acid (PFUnDA) accelerates insulitis development in a mouse model of type 1 diabetes

Perfluoralkylated substances (PFAS) are classified as persistent, bioaccumulative and toxic substances and are widespread environmental contaminants. Humans are exposed through food, drinking water and air. We have previously reported that bisphenol A accelerates spontaneous diabetes development in non-obese diabetic (NOD) mice and observed in the present study that perfluoroundecanoic acid, PFUnDA, increased insulitis development, a prerequisite for diabetes development in NOD mice. We exposed NOD mice to PFUnDA in drinking water (3, 30 and 300 μg/l) at mating, during gestation and lactation and until 30 weeks of age. After 300 μg/l PFUnDA exposure, we report (i) increased pancreatic insulitis, (ii) increased number of apoptotic cells in pancreatic islets prior to insulitis and (iii) decreased phagocytosis in peritoneal macrophages. There was also a trend of decreased number of tissue resident macrophages in pancreatic islets prior to insulitis after exposure to 300 μg/l, and altered cytokine secretion in activated splenocytes after exposure to 3 μg/l PFUnDA. Although insulitis is a prerequisite for autoimmune diabetes, the accelerated insulitis was not associated with accelerated diabetes development. Instead, the incidence of diabetes tended to be reduced in the animals exposed to 3 and 30 μg/l PFUnDA, suggesting a non-monotonic dose response. The effects of PFUnDA exposure on increased apoptosis in pancreas and reduced macrophage function as well as accelerated insulitis development in NOD mice, may also be relevant for human insulitis. Further observational autoimmune diabetes clinical cohort studies and animal experiments for PFUnDA as well as other PFASs are therefore encouraged.

Safety assessment in rats and dogs of Acoustic Cluster Therapy, a novel concept for ultrasound mediated, targeted drug delivery

Acoustic Cluster Therapy (ACT) represents a novel concept for targeted drug delivery. Ultrasound is applied to activate intravenously administered free‐flowing clusters of microbubbles and microdroplets within the target pathology, depositing 20–30 μm large bubbles in the microvasculature for 5–10 min. Further application of ultrasound induces biomechanical effects which increase vascular permeability and enhance localized extravasation of coadministered drugs. Herein we report investigations done to assess the preclinical safety of ACT, using doses up to 1 mL/kg (3 μL perfluoromethyl‐cyclopentane/kg). In dogs, half the animals were exposed to ultrasound activation in the heart for 1 min, no ultrasound was applied in the other half. Posttreatment observation time was 24 h. Clinical signs, ophthalmoscopy, clinical pathology, macro‐, and microscopy were used as endpoints. No differences between groups with and without ultrasound activation were observed. Short‐lasting leukopenia and thrombocytopenia, possibly secondary to a slight and short‐lasting increase in plasma histamine and complement split products, were the only effects noted. In rats ACT was activated in the liver for 5 min. Histopathology and clinical chemistry parameters remained unchanged. Lastly, rats were treated with ACT activated in the heart and thereafter placed on a rotarod for evaluation of motor coordination. No differences were observed between animals treated with ACT and controls. In conclusion, ACT appeared safe at dose‐levels up to 1 mL/kg and with activation either in the heart or the liver. These results, together with positive efficacy data upon coinjection with cytotoxic compounds encourage further preclinical safety studies with the objective of entering subsequent clinical trials.

Early life exposure to air pollution particulate matter (PM) as risk factor for attention deficit/hyperactivity disorder (ADHD): Need for novel strategies for mechanisms and causalities

&NA; Epidemiological studies have demonstrated that air pollution particulate matter (PM) and adsorbed toxicants (organic compounds and trace metals) may affect child development already in utero. Recent studies have also indicated that PM may be a risk factor for neurodevelopmental disorders (NDDs). A pattern of increasing prevalence of attention deficit/hyperactivity disorder (ADHD) has been suggested to partly be linked to environmental pollutants exposure, including PM. Epidemiological studies suggest associations between pre‐ or postnatal exposure to air pollution components and ADHD symptoms. However, many studies are cross‐sectional without possibility to reveal causality. Cohort studies are often small with poor exposure characterization, and confounded by traffic noise and socioeconomic factors, possibly overestimating the study associations. Furthermore, the mechanistic knowledge how exposure to PM during early brain development may contribute to increased risk of ADHD symptoms or cognitive deficits is limited. The closure of this knowledge gap requires the combined use of well‐designed longitudinal cohort studies, supported by mechanistic in vitro studies. As ADHD has profound consequences for the children affected and their families, the identification of preventable risk factors such as air pollution exposure should be of high priority. HighlightsAir pollution particles (PM) may lead to developmental neurotoxicity (DNT).Air pollution components have been associated with increased risk of ADHD.Current lack of longitudinal cohort studies covering pre‐ and postnatal PM exposure.Need for mechanistic studies on receptor/signalling pathways linked to DNT and ADHD.High throughput screening and advanced cell models are promising for DNT testing.

Hypotheses and evidence related to intense sweeteners and effects on appetite and body weight changes: A scoping review of reviews

Observed associations between consumption of diet foods and obesity have sparked controversy over whether intense sweeteners may promote weight gain, despite their negligible energy contribution. We conducted a scoping review of reviews, to obtain an overview of hypotheses, research approaches and features of the evidence on intense sweeteners’ potential relationships to appetite and weight changes. We searched for reviews of the scientific literature published from 2006 to May 2017. Two reviewers independently assessed title and abstracts, and full text publications. Arksey and O’Malley’s framework for scoping reviews guided the process. We extracted and charted data on characteristics of the reviews and the evidence presented. The 40 included reviews present hypotheses both on how intense sweeteners can reduce or maintain body weight and on how these can promote weight gain. We classified only five publications as systematic reviews; another nine presented some systematic approaches, while 26 reviews did not describe criteria for selecting or assessing the primary studies. Evidence was often presented for intense sweeteners as a group or unspecified, and against several comparators (e.g. sugar, water, placebo, intake levels) with limited discussion on the interpretation of different combinations. Apart from the observational studies, the presented primary evidence in humans is dominated by small studies with short follow-up—considered insufficient to assess weight change. Systematic reviews of animal studies are lacking in this topic area. The systematic evidence only partly explore forwarded hypotheses found in the literature. Primary studies in humans seem to be available for systematic exploration of some hypotheses, but long-term experimental studies in humans appear sparse. With few exceptions, the reviews on intense sweeteners and weight change underuse systematic methodology, and thus, the available evidence. Further studies and systematic reviews should be explicit about the hypothesis explored and elucidate possible underlying mechanisms.

Allergen Immunization Induces Major Changes in Microbiota Composition and Short-Chain Fatty Acid Production in Different Gut Segments in a Mouse Model of Lupine Food Allergy

Background: The incidence of food allergies in western countries has increased in recent decades. Objectives: To study the association between gut bacterial microbiota composition, short-chain fatty acids (SCFAs) and food allergy in a mouse model. Methods: After oral immunizations with the human food allergen lupine with the adjuvant cholera toxin (CT) (or buffer in controls), sensitization and anaphylactic responses were determined. Gastrointestinal content was collected from the distal ileum, cecum, colon, and fecal pellets, and the bacterial diversity and composition was determined by deep sequencing of the 16S rRNA gene. SCFAs in gastrointestinal content supernatants were determined by gas chromatography. Results: The microbiota signatures were profoundly affected by allergen immunization. Ten operational taxonomic units (OTUs) were significantly different between immunized and control animals for at least one of the intestinal segments; eight of these OTUs belonged to the Clostridia class. Although consistent across all four gut segments, the colon showed the highest number of OTUs significantly associated with allergic immunization. SCFA levels in the cecum were also altered by immunization. Conclusions: Allergen immunization with CT in the present food allergy model induced profound changes in the microbiome composition and SCFA production. The result suggests that the colon may be the most sensitive gut segment for investigating changes in the gut microbiome.

Decreased macrophage phagocytic function due to xenobiotic exposures in vitro, difference in sensitivity between various macrophage models

Both autoimmune disease prevalence and exposure to immunotoxic chemicals have increased the last decades. As a first screening of immunotoxic chemicals possibly affecting development of autoimmunity through attenuated macrophage function, we demonstrate a promising model measuring macrophage function in isolated peritoneal macrophages (PCM) from Wistar rats and C57Bl/6 mice. Immunotoxic effects of bisphenol A (BPA) and a selection of perfluoroalkyl acids (PFAAs) were analysed in vitro assessing phagocytic function of macrophages from different sources. Phagocytosis was reduced in PCM of C57Bl/6 mice and Wistar rats after BPA and perfluoroundecanoic acid (PFUnDA) exposure, but not in macrophages derived from human and rat monocyte derived macrophages (MDM). On the other hand, in vitro exposure to mixtures of persistent organic pollutants (POPs) showed similar reductions in rat PCM and rat and human MDM phagocytosis. Reduced phagocytosis was partly due to cytotoxicity. PCM isolated from non-obese diabetic (NOD) mice, interleukin 1α/β knockout (IL-1KO) mice and new-born rats were less sensitive to the xenobiotics than PCM from adult wild type rodents. Finally, in vivo studies with NOD mice verified that POP exposure also decreased the number of pancreatic macrophages in pancreatic islets, reflecting early signs of autoimmunity development, similarly as previously described for BPA.

Genetically and dietary induced obesity associate differently with gut microbiota in a murine intestinal tumorigenesis model

Background: Overweight and obesity are risk factors for human colorectal cancer. Growing evidence suggests that the gut microbiome affects both obesity and cancer. In this study, we examined how the murine microbiota composition correlated with obesity, intestinal tumorigenesis, glucose regulation, and inflammation. Materials and Methods: We used 16S ribosomal RNA gene analyses of feces and data obtained from a double-mutant mouse model; multiple intestinal neoplasia (Min), mice, which spontaneously develop intestinal tumors, crossed with obesity (ob), mice, which develop obesity, fed 10% or 45% fat diet. Results: We found that diet and genotypes imposed a major impact on the gut microbiota composition. Likewise, we found strong associations of the microbiota composition with obesity, number of small intestinal tumors, and blood glucose levels. Specifically, bacteria related to Clostridium perfringens and Lactobacillus showed strong positive associations with both dietary induced and genetically induced obesity, while Bacteroidales showed strong negative associations. Representatives of Lachnospiraceae and Peptostreptococcaceae only showed significant negative associations with genetically induced obesity and no associations with dietary induced obesity. Conclusions: We found complex associations between the microbiota and genetic background, diet, obesity, glucose levels, inflammation, and intestinal tumorigenesis. This could contribute to the lack of consensus between the results in previous studies regarding correlations of microbiota with obesity and cancer.