Repeated Exposure Enhanced Toxicity of Clarithromycin on Microcystis aeruginosa Versus Single Exposure through Photosynthesis, Oxidative Stress, and Energy Metabolism Shift.

Antibiotics are being increasingly detected in aquatic environments, and their potential ecological risk is of great concern. However, most antibiotic toxicity studies involve single-exposure experiments. Herein, we studied the effects and mechanisms of repeated versus single clarithromycin (CLA) exposure on Microcystis aeruginosa. The 96 h effective concentration of CLA was 13.37 µg/L upon single exposure but it reduced to 6.90 µg/L upon repeated exposure. Single-exposure CLA inhibited algal photosynthesis by disrupting energy absorption, dissipation and trapping, reaction center activation, and electron transport, thereby inducing oxidative stress and ultrastructural damage. In addition, CLA upregulated glycolysis, pyruvate metabolism, and the tricarboxylic acid cycle. Repeated exposure caused stronger inhibition of algal growth via altering photosynthetic pigments, reaction center subunits biosynthesis, and electron transport, thereby inducing more substantial oxidative damage. Furthermore, repeated exposure reduced carbohydrate utilization by blocking the pentose phosphate pathway, consequently altering the characteristics of extracellular polymeric substances and eventually impairing the defense mechanisms of M. aeruginosa. Risk quotients calculated from repeated exposure were higher than 1, indicating significant ecological risks. This study elucidated the strong influence of repeated antibiotic exposure on algae, providing new insight into antibiotic risk assessment.

Effect of cell treatment procedures on in vitro genotoxicity assessment.

So far, the majority of in vitro toxicological experiments are conducted after an acute 24 h treatment that does not represent a realistic human chemical exposure. Recently, new in vitro approaches have been proposed to study the chemical toxicological effect over several days in order to be more predictive of a representative exposure scenario. In this study, we investigated the genotoxic potential of chemicals (direct or bioactived clastogen, aneugen and apoptotic inducer) with the γH2AX and pH3 biomarkers, in the human liver-derived HepaRP cell line. We used different treatment durations, with or without a three-day recovery stage (release period), before genotoxicity measurement. Data were analysed with the Benchmark Dose approach. We observed that the detection of clastogenic compounds (notably for DNA damaging agents) was more sensitive after three days of repeated treatment compared to one or three treatments over 24 h. In contrast, aneugenic chemicals were detected as genotoxic in a similar manner whether after a 24 h exposure or a three-day repeated treatment. Globally, the release period decreases the genotoxicity measurement substantially. For DNA damaging agents, after high concentration treatments, γH2AX induction was always observed after a three-day release period. In contrast, for DNA topoisomerase inhibitors, no effect could be observed after the release period. In conclusion, in the HepaRP cell line, there are some important differences between a one-day acute and a three-day repeated treatment protocol, indicating that different cell treatment procedures may differentiate chemical genotoxic mechanisms of action more efficiently.

What's cooking, if not meat? Effects of repeated home-use, recipe inspiration and meal context on perception of plant-based meat analogues.

Plant-based meat analogues (PBMA) may help consumers in shifting towards more plant-based diets, but PBMA are not widely used yet, and little is known about their longer-term acceptance. This study investigated whether consumer acceptance of PBMA changed with repeated home-use, and whether providing recipe suggestions in the form of meal boxes could influence PBMA acceptance. To this end, Dutch regular meat eaters (n = 61) prepared, consumed and evaluated two meals (one from a meal box, one self-created) with PBMA (PB mince and PB chicken, counterbalanced across meal types) per week at home for four weeks. As a secondary objective, potential longer-term effects of repeated home-use of PBMA on meat (analogue) consumption habits and attitudes (e.g. motives for choosing PBMA, attitudes toward eating less meat) were assessed in a pre-vs post-intervention survey. Responses were compared with a control group of consumers not participating in the home-use study (n = 179). Results provided no evidence that PBMA liking changed with repeated home-use, nor that the provision of meal boxes increased liking of PBMA. Instead, PBMA liking was strongly influenced by the meal context, which may have overruled potential effects of repeated exposure. Findings from the pre- vs. post-intervention survey suggest that repeated exposure may stimulate longer-term consumption of PBMA, although more seems needed to bring about a structural shift toward a less animal-based consumption pattern. Future research should investigate whether more sophisticated recipes that provide a suitable meal context for PBMA and elevate consumers' meal experiences may improve PBMA acceptance and facilitate the transition toward more sustainable diets.

Low-concentration repeated exposure and high-concentration single exposure of cyanamide suppressed the growth of redroot pigweed in the alfalfa field.

The inclination toward natural products has led to the onset of the discovery of new bioactive metabolites that could be targeted for specific therapeutic or agronomic applications. Despite increasing knowledge coming to light of plant-derived materials as leads for new herbicides, relatively little is known about the mode of action on herbicide-resistant weeds. Cyanamide (CA) is a naturally occurring herbicide synthesized by hairy vetch (Vicia villosa Roth.). However, it has not been experimentally verified whether CA suppresses target plants via sustained discharge at low concentrations, as is often the case with most plant-derived materials. This study aimed to detect the toxicity and the mode of action of CA to alfalfa (Medicago sativa L.) and redroot pigweed (Amaranthus retroflexus L.). The toxicity of CA toward the alfalfa and redroot pigweed by three different exposure patterns was compared: low-concentration repeated exposure with 0.3 g/L CA (LRE), high-concentration single exposure with 1.2 g/L CA (HSE), and distilled water spray as control. The results showed that CA had a stronger inhibitory effect on redroot pigweed growth compared to alfalfa under both LRE and HSE exposure modes, with leaves gradually turning yellow and finally wilting. Beyond that, field trials were conducted to corroborate the toxicity of CA to alfalfa and redroot pigweed. The results have also shown that CA could inhibit the growth of redroot pigweed without significant adverse effects on alfalfa. The outcomes concerning electrolyte permeability, root activity, and malondialdehyde (MDA) content indicated that CA suppressed the growth of redroot pigweed by interfering with the structure of the cell membrane and impacting cellular osmotic potential. CA could destroy the cell membrane structure to inhibit the growth of the redroot pigweed by both LRE and HSE exposure modes, which provides a theoretical basis for preventing and controlling redroot pigweed in alfalfa fields.

Fifteen day repeat air: liquid Interface air-only exposures can cause respiratory epithelium injury in MucilAir™ nasal respiratory epithelial cells that parallels chemically induced cytotoxicity.

New Approach Methodologies (NAMs) are being widely used to reduce, refine, and replace, animal use in studying toxicology. For respiratory toxicology, this includes in silico and in vitro alternatives using air:liquid interface (ALI) exposures to replace traditional in vivo inhalation studies. In previous studies using 1,3-dichloropropene (1,3-DCP), a 5-day 4 h repeat exposures of MucilAir™ nasal cell culture models caused, dose-dependent cytotoxicity, depletion of GSH, changes in differential gene expression and histopathological transitions in cellular morphology from pseudostratified columnar epithelium to squamous epithelium. In this report we attempted to extend these studies using 15-day 1,3-DCP 4 h exposures to using MucilAir™ nasal cultures as outlined by an US EPA recent task order (US EPA 2023). For the 15-day repeat exposure, there were severe histopathologic changes in the MucilAir™ nasal mock-treatment (air-only) VITROCELL® chamber controls compared to incubator controls preventing any further analysis. The histopathological transitions in cellular morphology from pseudostratified columnar epithelium to squamous epithelium observed in the air only control in this study and previously with 1,3-DCP in MucilAir™ nasal cultures is also a hallmark of chemically induced cytotoxic responses in vivo in the respiratory tract. Histopathology assessments of 3D respiratory tract models used in ALI exposures can provide the linkage between in vitro to in vivo outcomes as part of the validation efforts of ALI use in regulatory toxicology. This report indicates that importance of histopathological assessments of incubator and mock-treatment (air-only) controls from each ALI exposure experiment along with exposed cell based model.

Preschool- and childcare center-based interventions to increase fruit and vegetable intake in preschool children in the United States: a systematic review of effectiveness and behavior change techniques.

BACKGROUND: Fruit and vegetable (FV) consumption in children in the United States (US) is very low. Adequate FV consumption is required for proper development during childhood, and dietary habits are established during preschool-age and tend to persist into adulthood. As most U.S. preschool-aged children attend childcare or preschool, this may be an opportune time and setting to conduct interventions to improve FV intake. These interventions should be based in theory and use behavior change techniques (BCTs) to explain mechanisms for expected change. To date, no published reviews have examined the effectiveness of childcare- or preschool-based FV interventions in preschoolers and their use of theoretical frameworks and BCTs. METHODS: This systematic review was completed adhering to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Inclusion criteria were randomized controlled trials (RCTs) published between 2012 and 2022 of interventions to improve diet or FV intake in preschoolers (aged 2-5 years) in childcare or preschool-settings. A search of four databases was conducted between in September 2022 using search terms pertaining to the study's primary aim (FV consumption), age group (preschool-aged), settings (US childcare or preschool settings), and study design (RCT). Additional criteria were objective measures of FV consumption or skin carotenoids, as a proxy for FV intake. Included studies were narratively synthesized based on intervention type, measured effect, and use of theory and BCTs. RESULTS: The search resulted in six studies that reported on nine interventions. Overall, six interventions increased FV intake, of which five used nutrition education and one manipulated the feeding environment. Among the three interventions with no measured effect, two manipulated the feeding environment and one used peer modeling. Effective studies used at least three BCTs, though no pattern was observed between use of theory or BCTs and intervention effect. CONCLUSIONS: While several studies have shown promising results, the limited number of studies identified in this review highlights key gaps in this field: there is a need for studies to test FV interventions in US childcare settings that use objective measures of FV intake, directly compare intervention components and BCTs, are theory-based, and assess long-term behavior change.

Promoting vegetable acceptance in toddlers using a contingency management program: A cluster randomised trial.

Children learn to accept novel foods by repeated exposure to these foods. In the current study, we investigated in toddlers whether a contingency management program (The Vegetable Box), comprising repeated vegetable taste exposure with contingent non-food rewards, is particularly potent at increasing recognition of and willingness to try vegetables. A total of 598 children (1-4 years old) recruited at 26 different day-care centres in the Netherlands took part. The day-care centres were randomly assigned to one of three conditions ('exposure/reward', 'exposure/no reward', or 'no exposure/no reward'). At the start and directly after the 3-month intervention period, all children were asked to identify various vegetables (recognition test; max score = 14) and whether they would like to taste and consume 1 or 2 bite-size pieces of tomato, cucumber, carrot, bell pepper, radish, and cauliflower (willingness to try test). Data were analysed with linear mixed-effects regression analyses (for recognition and willingness to try separately) with condition and time as independent variables, and adjusting for day-care centre clustering. Vegetable recognition significantly increased in both the 'exposure/reward' and the 'exposure/no reward' group, relative to the 'no exposure/no reward' control group. The willingness to try vegetables only increased significantly in the 'exposure/reward' group. Offering vegetables to children at day-care centres significantly increased toddlers' ability to identify various vegetables, but rewards contingent upon tasting vegetables appear particularly effective to also increase children's willingness to try (i.e., taste and consume) different vegetables. This result corroborates and strengthens previous findings demonstrating the efficacy of similar reward-based programs.

Role-model, reoffer, reward: A thematic analysis and TDF mapping of influences on families' use of evidence-based vegetable feeding practices.

Children's vegetable intake is low, despite benefits for immediate and long-term health. Repeatedly reoffering vegetables, role-modelling consumption, and offering non-food rewards effectively increase children's vegetable acceptance and intake. However, a number of barriers prevent families from reoffering previously-rejected vegetables. This study used the Theoretical Domains Framework (TDF) and the COM-B model of behaviour to explore barriers and enablers to reoffering, role-modelling and offering non-food rewards among parents of 2-4-year-old children. Twenty-five semi-structured interviews were conducted, from which eleven core inductive themes were generated: 'Child factors', 'Eating beliefs', 'Effectiveness beliefs', 'Past experience', 'Current family behaviours', 'Harms', 'Knowledge', 'Need for change', 'Parent effort', 'Parent values' and 'Practical issues'. The codes underpinning these themes were inductively mapped to 11 of the 14 TDF domains, and five of the six COM-B components. Previously-reported influences on families' vegetable feeding practices were confirmed, including concerns about child rejection of foods/meals, cost of vegetables, and food waste. Novel findings included some parents' perceptions that these practices are pressurising, and that certain beliefs/knowledge about children's eating behaviour can provide a "protective mindset" that supports families' perseverance with reoffering over time. Future interventions should be tailored to better reflect the diversity of needs and previous experiences of feeding that families have, with some families likely to find that troubleshooting and further signposting is appropriate for their needs while others might benefit from more persuasive and educational approaches. The mapping of codes to the TDF and COM-B will facilitate the identification of appropriate intervention functions and behaviour change techniques when designing new interventions to support families with increasing their children's vegetable intake.

Responses of cyanobacterium Microcystis aeruginosa under single and repeated ofloxacin exposure.

Antibiotics are omnipresent and pseudo-persistent in the environment. Yet, their potential ecological risks under repeated exposure, which is more environmentally relevant, are understudied. Therefore, this study used ofloxacin (OFL) as the probe chemical to investigate the toxic effects of different exposure scenarios-single dose of high concentration (4.0 µg/L) and multiple additions of low concentrations-towards the cyanobacterium Microcystis aeruginosa. Flow cytometry was employed to measure a collection of biomarkers, including endpoints related with biomass, single cell properties and physiological status. Results showed that the single dose of the highest OFL level inhibited cellular growth, chl-a content and cell size of M. aeruginosa. In contrast, OFL induced stronger chl-a autofluorescence and higher doses tended to have more remarkable effects. Repeated low OFL doses can more significantly increase the metabolic activity of M. aeruginosa than a single high dose. Viability and cytoplasmic membrane were not affected by OFL exposure. Oxidative stress was observed for the different exposure scenarios, with fluctuating responses. This study demonstrated the different physiological responses of M. aeruginosa under different OFL exposure scenarios, providing novel insights into the toxicity of antibiotics under repeated exposure.

The Baby's First Bites RCT: Evaluating a Vegetable-Exposure and a Sensitive-Feeding Intervention in Terms of Child Health Outcomes and Maternal Feeding Behavior During Toddlerhood.

BACKGROUND: Parenting interventions during the first years of life on what and/or how to feed infants during complementary feeding can promote healthy eating habits. OBJECTIVES: An intervention promoting repeated exposure to a variety of vegetables [repeated vegetable exposure (RVE); what] and an intervention promoting responding sensitively to child signals during mealtime [video-feedback intervention to promote positive parenting-feeding infants (VIPP-FI); how] were compared, separately and combined (COMBI), with an attention control condition (AC). Primary outcomes were vegetable consumption and self-regulation of energy intake; secondary outcomes were child anthropometrics and maternal feeding practices (sensitive feeding, pressure to eat). METHODS: Our 4-arm randomized controlled trial included 246 first-time Dutch mothers and their infants. Interventions started when infants were 4-6 mo old and ended at age 16 mo. The present study evaluated effects at 18 (t18) and 24 (t24) mo of age. Vegetable acceptance was assessed using three 24-h dietary recalls, self-regulation of energy intake by an eating-in-the-absence-of-hunger experiment and mother-report, and maternal feeding behavior by observation and mother-report. RESULTS: Linear mixed model and ANOVA analyses revealed no follow-up group differences regarding child vegetable intake or self-regulatory behavior. The proportion of children with overweight was significantly lower in the COMBI group, compared with the VIPP-FI group at t18 (2% compared with 16%), and with the AC group at t24 (7% compared with 20%), although this finding needs to be interpreted cautiously due to the small number of infants with overweight and nonsignificant effects on the continuous BMI z-score measure (P values: 0.29-0.82). Finally, more sensitive feeding behavior and less pressure to eat was found in the VIPP-FI and COMBI groups, compared with the RVE and AC groups, mostly at t18 (significant effect sizes: d = 0.23-0.64). CONCLUSIONS: Interventions were not effective in increasing vegetable intake or self-regulation of energy intake. Future research might usefully focus on risk groups such as families who already experience problems around feeding.This trial is registered at clinicaltrials.gov as NCT03348176.

Chronic effects of two rutile TiO2 nanomaterials in human intestinal and hepatic cell lines.

BACKGROUND: TiO2 nanomaterials (NMs) are present in a variety of food and personal hygiene products, and consumers are exposed daily to these NMs through oral exposition. While the bulk of ingested TiO2 NMs are eliminated rapidly in stool, a fraction is able to cross the intestinal epithelial barrier and enter systemic circulation from where NMs can be distributed to tissues, primarily liver and spleen. Daily exposure to TiO2 NMs, in combination with a slow rate of elimination from tissues, results in their accumulation within different tissues. Considerable evidence suggests that following oral exposure to TiO2 NMs, the presence of NMs in tissues is associated with a number of adverse effects, both in intestine and liver. Although numerous studies have been performed in vitro investigating the acute effects of TiO2 NMs in intestinal and hepatic cell models, considerably less is known about the effect of repeated exposure on these models. In this study, we investigated the cytotoxic effects of repeated exposure of relevant models of intestine and liver to two TiO2 NMs differing in hydrophobicity for 24 h, 1 week and 2 weeks at concentrations ranging from 0.3 to 80 µg/cm2. To study the persistence of these two NMs in cells, we included a 1-week recovery period following 24 h and 1-week treatments. Cellular uptake by TEM and ToF-SIMS analyses, as well as the viability and pro-inflammatory response were evaluated. Changes in the membrane composition in Caco-2 and HepaRG cells treated with TiO2 NMs for up to 2 weeks were also studied. RESULTS: Despite the uptake of NM-103 and NM-104 in cells, no significant cytotoxic effects were observed in either Caco-2 or HepaRG cells treated for up to 2 weeks at NM concentrations up to 80 µg/cm2. In addition, no significant effects on IL-8 secretion were observed. However, significant changes in membrane composition were observed in both cell lines. Interestingly, while most of these phospholipid modifications were reversed following a 1-week recovery, others were not affected by the recovery period. CONCLUSION: These findings indicate that although no clear effects on cytotoxicity were observed following repeated exposure of differentiated Caco-2 and HepaRG cells to TiO2 NMs, subtle effects on membrane composition could induce potential adverse effects in the long-term.

Pharmacokinetics and toxicity evaluation following oral exposure to bisphenol F.

Bisphenol F is a substitute material for bisphenol A and is widely used in household products as a raw material for polycarbonate resin, epoxy resin, and plastic reinforcement. It is known to be mainly used in food containers, thermal paper for receipts, and coatings for water pipes. In some countries, bisphenol F has been detected in drinking water and human urine samples. However, due to the lack of safety evaluation data on bisphenol F, it is difficult to establish appropriate guidelines for the proper use of the substance, and social anxiety is increasing accordingly. This study investigated the use, exposure route, and distribution flow of bisphenol F, a household chemical. To determine the no-observed-adverse-effect level (NOAEL) and target organ of bisphenol F after exposure, a single-dose oral toxicity, dose-range finding (28 day oral), repeated dose toxicity (90 day oral), and genotoxicity (reverse mutation, chromosomal abnormality, in vivo micronucleus test) tests were performed. The pharmacokinetic profile was also obtained. The test results are as follows: in the pharmacokinetic study, it was confirmed that single oral exposure to BPF resulted in systemic exposure; in single oral dose toxicity test, the approximate lethal dose was found to be 4000 mg/kg and confusion and convulsion was shown in the test animals; NOAEL was determined to be 2 mg/kg/day for male and 5 mg/kg/day for female, and the no-observed-effect level (NOEL) was determined to be 2 mg/kg/day for males and 1 mg/kg/day for females, and the target organ was the small intestine; genotoxicity tests confirmed that BPF does not induce genotoxicity.

Oral exposure to aluminum chloride for 28 days suppresses neural stem cell proliferation and increases mature granule cells in adult hippocampal neurogenesis of young-adult rats.

Aluminum (Al), a common light metal, affects the developing nervous system. Developmental exposure to Al chloride (AlCl3 ) induces aberrant neurogenesis by targeting neural stem cells (NSCs) and/or neural progenitor cells (NPCs) in the dentate gyrus (DG) of rats and mice. To investigate whether hippocampal neurogenesis is similarly affected by AlCl3 exposure in a general toxicity study, AlCl3 was orally administered to 5-week-old Sprague Dawley rats at dosages of 0, 4000, or 8000 ppm in drinking water for 28 days. AlCl3 downregulated Sox2 transcript level in the DG at the highest dosage and produced a dose-dependent decrease of SOX2+ cells without altering numbers of GFAP+ or TBR2+ cells in the subgranular zone, suggesting that AlCl3 decreases Type 2a NPCs. High-dose exposure downregulated Pcna, upregulated Pvalb, and altered expression of genes suggestive of oxidative stress induction (upregulation of Nos2 and downregulation of antioxidant enzyme genes), indicating suppressed proliferation and differentiation of Type 1 NSCs. AlCl3 doses also increased mature granule cells in the DG. Upregulation of Reln may have contributed to an increase of granule cells to compensate for the decrease of Type 2a NPCs. Moreover, upregulation of Calb2, Gria2, Mapk3, and Tgfb3, as well as increased numbers of activated astrocytes in the DG hilus, may represent ameliorating responses against suppressed neurogenesis. These results suggest that 28-day exposure of young-adult rats to AlCl3 differentially targeted NPCs and mature granule cells in hippocampal neurogenesis, yielding a different pattern of disrupted neurogenesis from developmental exposure.

The effect of parental food neophobia on children's fruit and vegetable consumption: A serial mediation model.

Children in the United States do not consume the recommended amounts of healthful foods such as fruits and vegetables. In order to investigate factors associated with children's fruit and vegetable consumption, we used serial mediation models based on a theoretical model proposed by Kaar et al., [Food Quality & Preference, 50, 57-64 (2016)] to assess the indirect effect of parental food neophobia (X) on children's consumption (Y) of fruits and vegetables through two serial mediators, variety of fruits and vegetables offered at home (variety offered; M1) and child food neophobia (M2). This was accomplished with a sample of 148 children between 5 and 10 years of age (Mage = 7.71 years, 44.7% female). We measured parental and child food neophobia and parents' reports of the frequency with which they regularly offered a variety of fruits and vegetables. In addition, children completed a laboratory task in which they were presented with four different fruits and vegetables and were asked to try the foods. Analyses revealed a significant indirect effect of parents' food neophobia on consumption of fruits and vegetables through variety offered (M1) and child food neophobia (M2). There was also a significant serial indirect effect of parent food neophobia on consumption through variety offered and child food neophobia. The results provide further evidence to support the contention that parent and child neophobia and the feeding environment exert a strong influence on children's fruit and vegetable consumption.

Effects of repeated exposure to 50 Hz electromagnetic field on breast cancer cells.

The extremely low frequency electromagnetic field (ELF-EMF) is emerging as a novel approach in cancer treatment. This study evaluated the impact of daily exposure to 50 Hz EMF on breast cancer cells in vitro. The MDA-MB-231 and MCF-7 cells were exposed to EMF (50 Hz 20 mT, for 3 hours per day for up to four days) and examined for cell vaibility. The effect of daily ELF-EMF exposure on cell cycle progression and cell death was further investigated. The result revealed that the consecutive exposure to 50 Hz EMF at 20 mT remarkably decreased the viability of MDA-MB-231 compared to the non-exposed group, while it had no significant effect on MCF-7 cells. The ELF-EMF exposure induced G1 phase arrest along with the increase in sub-G1 cell population in MDA-MB-231. Moreover, repeated exposure to 50 Hz EMF promoted cell cycle progression in MCF-7 by increasing the percentage of cells in the S phase. The fluorescent staining revealed that daily exposure of ELF-EMF induced apoptotic cell death in MDA-MB-231, but no morphological change was observed in MCF-7 cells. The results showed that repeated daily exposure to 50 Hz EMF exhibited anti-proliferative activity against invasive breast cancer cells by impairing cell cycle progression and inducing cell death.

Infant and Toddler Consumption of Sweetened and Unsweetened Lipid Nutrient Supplements After 2-Week Home Repeated Exposures.

BACKGROUND: Small-quantity lipid-based nutrient supplements (SQ-LNS) are designed to address undernutrition during the complementary feeding period. SQ-LNS contains added sugars, but limited research has assessed whether infants' acceptance varies between versions with and without sugars. OBJECTIVES: Our objective was to examine the effects of repeated exposure on children's acceptance of sweetened and unsweetened SQ-LNS. We aimed to understand caregivers' perceptions of children's liking of the 2 SQ-LNS versions and their influences on infant acceptance of SQ-LNS. METHODS: Caregivers (86% non-Hispanic White) and children (7-24 mo), participated in a randomized, 2-week home-exposure study and baseline and post-home exposure assessments. Children were randomized to receive sweetened or unsweetened SQ-LNS versions, mixed with infant oatmeal. At in-person visits, caregivers fed both SQ-LNS versions to children and rated their child's liking for each. Caregivers fed the SQ-LNS version to which their child was randomized until the child refused to eat more. Acceptance was measured as total grams consumed. Mixed-effects linear models tested the change in SQ-LNS consumed between baseline and postexposure by the SQ-LNS version and number of home exposures. Covariates included the amount of SQ-LNS consumed at baseline, child BMI z-score, child age, and breastfeeding experience. RESULTS: Children's acceptance of both SQ-LNS versions increased from baseline to postexposure (ß, 0.71 g; 95% CI: 0.54-0.89 g; P = 0.04), regardless of SQ-LNS version (P = 0.88) or number of home exposures (P = 0.55). Caregivers rated children's liking of unsweetened SQ-LNS higher at baseline (P = 0.02). Children with lower liking ratings at baseline showed the greatest increases in acceptance between baseline and postexposure (P = 0.01). CONCLUSIONS: Children's acceptance of SQ-LNS increased with repeated exposure, whether offered the sweetened or unsweetened version, providing preliminary support that adding sugar to SQ-LNS may not improve acceptance in young children. Children who initially like the supplement less may need repeated experience to learn to accept SQ-LNS. This trial was registered at clinicaltrials.gov as NCT04544332.

Toxicological responses of BEAS-2B cells to repeated exposures to benzene, toluene, m-xylene, and mesitylene using air-liquid interface method.

In order to reduce exposure to toxic chemicals, the European REACH regulation (1907/2006) recommends substituting toxic molecules with compounds that are less harmful to human health and the environment. Toluene is one of the most frequently used solvents in industries despite its toxicity. The objective of this study is to better understand and compare the toxicity of toluene and its homologues in a bronchial cell model. Thus, human bronchial BEAS-2B cells were exposed to steams of toluene, m-xylene, mesitylene (1,3,5-trimethylbenzene), and benzene (20 and 100 ppm). Exposure was carried out using an air-liquid interface (ALI) system (Vitrocell) during 1 h/day for 1, 3, or 5 days. Cytotoxicity, xenobiotic metabolism enzyme gene expression, and inflammatory response were evaluated following cell exposures. BEAS-2B cell exposure to toluene and its homologues revealed the involvement of major (CYP2E1) and minor metabolic pathways (CYP1A1). A late induction of genes (EPHX1, DHDH, ALDH2, and ALDH3B1) was measured from Day 3 and can be linked to the formation of metabolites. An increase in the secretion level of inflammatory markers (TNF-α, IL-6, IL-8, MCP-1, and GM-CSF) was also observed. In parallel, regulation between inflammatory mediators and the expression of transmembrane glycoprotein mucin MUC1 was also studied. This in vitro approach with ALI system points out the relevance of conducting repeated exposures to detect potential late effects. The difference recorded after cell exposure to toluene and its homologues highlights the importance of substitution principle.

Developmental exposure window influences silver toxicity but does not affect the susceptibility to subsequent exposures in zebrafish embryos.

Silver is a non-essential, toxic metal widespread in freshwaters and capable of causing adverse effects to wildlife. Its toxic effects have been studied in detail but less is known about how sensitivity varies during development and whether pre-exposures affect tolerance upon re-exposure. We address these knowledge gaps using the zebrafish embryo (Danio rerio) model to investigate whether exposures encompassing stages of development prior to mid-blastula transition, when chorion hardening and epigenetic reprogramming occur, result in greater toxicity compared to those initiated after this period. We conducted exposures to silver initiated at 0.5 h post fertilisation (hpf) and 4 hpf to determine if toxicity differed. In parallel, we exposed embryos to the methylation inhibitor 5-azacytidine as a positive control. Toxicity increased when exposures started from 0.5 hpf compared to 4 hpf and LC50 were significantly lower by 1.2 and 7.6 times for silver and 5-azacyitidine, respectively. We then investigated whether pre-exposure to silver during early development (from 0.5 or 4 hpf) affected the outcome of subsequent exposures during the larvae stage, and found no alterations in toxicity compared to naïve larvae. Together, these data demonstrate that during early development zebrafish embryos are more sensitive to silver when experiments are initiated at the one-cell stage, but that pre-exposures do not influence the outcome of subsequent exposures, suggesting that no long-lasting memory capable of influencing future susceptibility was maintained under our experimental conditions. The finding that toxicity is greater for exposures initiated at the one-cell stage has implications for designing testing systems to assess chemical toxicity.

Latency estimation for chronic disease risk: a damped exponential weighting model.

Identifying the susceptible period when environmental factors affect disease risk is essential for understanding disease etiology. Most existing epidemiologic studies use oversimplified summaries of time-dependent exposures such as baseline or most current exposure, or the cumulative average of exposure over available follow-up periods. In this paper, we introduce a damped exponential weighting model for estimating optimal exposure weights for different time intervals. This model can accommodate flexible patterns of weights and can be fit using standard software. We applied the model to assess the latency of BMI and alcohol for post-menopausal breast cancer based on 30-year exposure history in the Nurses' Health Study. We have also performed a simulation study to assess the validity of the proposed hypothesis testing and estimation procedures in realistic conditions. We found that the type I error is close to 0.05; the bias in our parameter estimates is low and the coverage probability of interval estimates is close to 0.95. For ER+/PR+ breast cancer we found that recent BMI was a more important predictor of risk than more distant BMI; for ER-/PR- breast cancer, no latency was found and risk was characterized by cumulative high levels of BMI over a long period of time. For alcohol intake, we saw a strong positive association with cumulative intake for ER+/PR+ breast cancer; no significant association was found for cumulative intake or for any latency measure of risk for ER-/PR- breast cancer. Our results underscore the value of an easy-to-implement approach to latency analysis of exposure profiles for chronic disease.

In vitro long-term repeated exposure and exposure switching of a novel tobacco vapor product in a human organotypic culture of bronchial epithelial cells.

Next-generation tobacco products and nicotine delivery systems such as heat-not-burn tobacco products and electronic cigarettes, the usage of which is expected to have a beneficial impact on public health, have gained popularity over the past decade. However, the risks associated with the long-term use of such products are still incompletely understood. Although the risks of these products should be clarified through epidemiological studies, such studies are normally performed based on each product category, not product-by-product. Therefore, investigation of the risk on a product-by-product basis is important to provide specific scientific evidence. In the current study, we performed the 40-day repeated exposure of in vitro human bronchial epithelial tissues to cigarette smoke (CS) or vapor from our proprietary novel tobacco vapor product (NTV). In addition, tissue samples exposed to CS were switched to NTV or CS exposure was stopped at 20 days to reflect a situation where smokers switched to NTV or ceased to smoke. All tissue samples were assessed in terms of toxicity, inflammation and transcriptomic alterations. Tissue samples switched to NTV and the cessation of exposure samples showed recovery from CS-induced damage although there was a time-course difference. Moreover, repeated exposure to NTV produced negligible effects on the tissue samples while CS produced cumulative effects. Our results suggest that the use of NTV, including switching to NTV from cigarette smoking, has fewer effects on bronchial epithelial tissues than continuing smoking.