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The isolation of stably transfected cell lines suitable for the manufacture of biotherapeutic protein products can be an arduous process relying on the identification of a high expressing clone; this frequently involves transgene amplification and maintenance of the clones' expression over at least 60 generations. Maintenance of expression, or cell line stability, is highly dependent upon the nature of the genomic environment at the site of transgene integration, where epigenetic mechanisms lead to variable expression and silencing in the vast majority of cases. We have assessed four chromatin function modifying elements (A2UCOE, MAR X_S29, STAR40 and cHS4) for their ability to negate chromatin insertion site position effects and their ability to express and maintain monoclonal antibody expression. Each element was analysed by insertion into different positions within a vector, either flanking or between heavy chain (HC) and light chain (LC) antibody expression cassettes. Our results clearly show that the A2UCOE is the most beneficial element in this system, with stable cell pools and clones increasing antibody yields 6.5-fold and 6.75-fold respectively. Stability analysis demonstrated that the reduction in antibody expression, seen with cells transfected with the control vector over 120 generations, was mitigated in the clones containing A2UCOE-augmented transgenes. Analysis also showed that the A2UCOE reduced the amount of transgene promoter DNA methylation, which contributed to the maintenance of starting levels of expression.

Glyphosate (GLY), the most widely used herbicide in the world, is frequently detected in various environmental matrices, including soil, the foundation of agriculture. In practice, more than 2000 GLY-based herbicide (GBH) products are used, consisting of one or more active ingredients (AIs) and so-called “inert” co-formulants that increase the efficacy of the AIs. However, the focus of ecotoxicological assessments is mainly on AIs, while organisms are exposed to complex pesticide formulations under real-world conditions. Overall, the effects on non-target organisms indicate a broad range of biochemical and physiological modes of action, which contrasts with the general assumption that herbicides are specific and act only on target plants. Both GLY alone and GBHs have unintended side-effects on many terrestrial organisms, including non-target plants, microorganisms, insects, spiders, or earthworms, as well as vertebrates such as amphibians, reptiles, or mammals. One of the triggering mechanisms for these effects is oxidative stress with consequences on biochemical parameters and DNA damage. In addition, disruptions of various physiological, behavioral and ecological processes have been reported. Most studies have examined the short-term effects of a single application of GLY/GBH to a single species. However, the agricultural practice of applying GBHs two to three times during a cultivation season over an extended period of time, the interactions with other pesticides and agrochemicals applied to the same field, and effects on ecological interactions within the field and landscape are rarely considered. In the vast majority of cases, the toxicity of GBHs exceeds the toxicity of GLY, demonstrating that supposedly inert co-formulants are either toxic in their own right or interact and add to the toxicity of AIs. The chemical diversity of different GBHs and the non-disclosure of the co-formulants make it difficult to attribute effects to specific chemical substances within a GBH. Moreover, impurities in GBHs (e.g., heavy metals such as arsenic, chromium, cobalt) pose additional environment and food safety risks. These impacts are even more critical because GBHs are so widely distributed worldwide and interact with other pollutants and environmental stressors. Based on the available literature on terrestrial ecotoxicity, and given the drastic decline in biodiversity, we conclude that the continued high use of GBHs, resulting in increased exposure and risk, cannot be considered ecologically sustainable.

Glyphosate (GLY), the most widely used herbicide active ingredient (AI) in the world, is frequently detected in aquatic environments where it can affect non-target organisms. Globally, more than 2000 commercial GLY-based herbicides (GBHs) are used to control weeds. Non-target organisms are exposed to complex pesticide formulations under real environmental conditions, but the co-formulants contained in GBHs are classified as so-called inert and inactive ingredients in terms of their biological effects. The main objective of this comprehensive review is to compile the results of aquatic ecotoxicological studies on the side-effects of GLY, GBHs, and their formulating agents. Based on the results demonstrated for a variety of plant and animal aquatic organisms, oxidative stress appears to be a major trigger for these adverse effects, affecting the integrity of DNA and other biochemical functions. Furthermore, there is evidence of impairment of various physiological and behavioral functions. Adverse effects of GLY and GBHs have been observed even at very low concentrations. There are also differences in the sensitivity of the aquatic organisms tested, even with similar lifestyles, habitats or identical taxa. The studies typically investigate the short-term effects of a single exposure to GLY/GBH on a single species, whilst in reality multiple applications of GBHs together with other pesticides are common during a cropping cycle. Moreover, the interactions between GLY/GBHs and other aquatic contaminants are rarely studied. Higher toxicity of GBHs compared to GLY alone has often been observed, demonstrating that co-formulants can be highly toxic on their own and markedly increase the toxicity of the GBH formulation. The possible impurities in GBHs, such as heavy metals, can cause additional problems for the environment and food safety. The widespread and massive use of GBHs leads to increased exposure and environmental hazards. In addition, the need for a revision of the risk assessment system is emphasized. According to the results of aquatic ecotoxicological studies, the current use and pollution of the aquatic environment by GLY/GBHs is highly problematic and cannot be considered environmentally sustainable. It is, therefore, necessary to at least tighten the permitted forms of use.

Pesticides are recognised as a key threat to pollinators, impacting their health in many ways. One route through which pesticides can affect pollinators like bumblebees is through the gut microbiome, with knock-on effects on their immune system and parasite resistance. We tested the impacts of a high acute oral dose of glyphosate on the gut microbiome of the buff tailed bumblebee ( Bombus terrestris ), and glyphosate’s interaction with the gut parasite ( Crithidia bombi ). We used a fully crossed design measuring bee mortality, parasite intensity and the bacterial composition in the gut microbiome estimated from the relative abundance of 16S rRNA amplicons. We found no impact of either glyphosate, C. bombi , or their combination on any metric, including bacterial composition. This result differs from studies on honeybees, which have consistently found an impact of glyphosate on gut bacterial composition. This is potentially explained by the use of an acute exposure, rather than a chronic exposure, and the difference in test species. Since A. mellifera is used as a model species to represent pollinators more broadly in risk assessment, our results highlight that caution is needed in extrapolating gut microbiome results from A. mellifera to other bee species.

Commercial pesticide formulations contain co-formulants, which are generally considered as having no toxic effects in mammals. This study aims to compare the toxicity of 8 major herbicide active ingredients–namely glyphosate, dicamba, 2,4-D, fluroxypyr, quizalofop-p-ethyl, pendimethalin, propyzamide and metazachlor–with a typical commercial formulation of each active ingredient. Cytotoxicity and oxidative stress capability was assessed in human hepatoma HepG2 cells. Using an MTT assay, formulations of glyphosate (Roundup Probio), fluroxypyr (Hurler), quizalofop-p-ethyl (Targa Super) and dicamba (Hunter) were more toxic than the active ingredient alone. Metazachlor and its formulation Sultan had similar cytotoxicity profiles. Cytotoxicity profiles were comparable in immortalised human fibroblasts. Toxilight necrosis assays showed the formulation of metazachlor (Sultan50C) resulted in significant membrane disruption compared to the active ingredient. Generation of reactive oxygen species was detected for glyphosate, fluroxypyr, pendimethalin, quizalofop-p-ethyl, the formulation of 2,4-D (Anti-Liserons), and dicamba and its formulation Hunter. Further testing of quizalofop-p-ethyl and its formulation Targa Super in the ToxTracker assay system revealed that both products induced oxidative stress and an unfolded protein response. In conclusion, these results show that most herbicide formulations tested in this study are more toxic than their active ingredients in human tissue culture cell model systems. The results add to a growing body of evidence, which implies that commercial herbicide formulations and not just their active ingredients should be evaluated in regulatory risk assessment of pesticides.

Objective The toxicology of herbicides, which are currently in use is under-explored. One highly used but under investigated herbicide is pendimethalin. Here we mined high-throughput data from the US National Toxicology Program (NTP) to identify whether pendimethalin possesses an estrogenic capability in human cells. We also evaluated effects of pendimethalin and its reference commercial formulated herbicide Stomp Aqua on the transcriptome profile of three human mammary epithelial cell lines, cancerous MCF-7 and non-cancerous MCF-10 A and MCF-12 A to see whether this compound could have endocrine disrupting effects and if co-formulants present in the commercial formulation could amplify its toxicity. Results The data mined from the US NTP database suggests that pendimethalin activates estrogen receptors at a concentration of approximately 10?M. MCF-7, MCF-10A and MCF-12A cells were exposed to 10 ?M pendimethalin and Stomp Aqua at an equivalent concentration. Transcriptome analysis showed changes in gene expression patterns implying that pendimethalin affected ubiquitin-mediated proteolysis and the function of the spliceosome. The formulated pendimethalin product Stomp Aqua gave comparable effects suggesting pendimethalin was responsible for the observed transcriptome alterations. Given the lack of information on the exposure to this pesticide, our study prompts the need for biomonitoring studies, especially under occupational use scenarios, to understand if low level exposure to pendimethalin could have endocrine disrupting effects on populations exposed to this compound. A deeper understanding of the exposure and mechanisms of action of this endocrine-disrupting pesticide is needed.

Body mass results from a complex interplay between genetics and environment. Previous studies of the genetic contribution to body mass have excluded repetitive regions due to the technical limitations of platforms used for population scale studies. Here we apply genome-wide approaches, identifying an association between adult body mass and the copy number (CN) of 47S-ribosomal DNA (rDNA). rDNA codes for the 18 S, 5.8 S and 28 S ribosomal RNA (rRNA) components of the ribosome. In mammals, there are hundreds of copies of these genes. Inter-individual variation in the rDNA CN has not previously been associated with a mammalian phenotype. Here, we show that rDNA CN variation associates with post-pubertal growth rate in rats and body mass index in adult humans. rDNA CN is not associated with rRNA transcription rates in adult tissues, suggesting the mechanistic link occurs earlier in development. This aligns with the observation that the association emerges by early adulthood. Many genetic variants have been associated with body size, but the contribution of copy number of rDNA is unknown. Here, the authors explore the association between rDNA copy number and body size in both rats and humans, finding that lower rDNA CN is associated with higher weight and BMI.

Misinformation has always existed, but it became a major preoccupation during the COVID-19 pandemic due to its ability to affect public health choices, decisions, and policy. In their article, “Misinformation in the media: Global coverage of GMOs 2019–2021” (GM Crops & Food, 17 Nov 2022), Mark Lynas et al. characterise critics of agricultural genetically modified organisms (GMOs) and their associated pesticides as purveyors of “misinformation”. They draw an equivalence between critics of agricultural GMOs and people who make false claims about climate change, COVID-19, and vaccines. We examined their main claims on these GMOs—for example, that there is a scientific consensus that they are safe for health and the environment—in the light of the scientific evidence and public discussion on this topic. We found that their claims are biased and misleading and ignore or omit crucial evidence. We conclude that based on the evidence provided, Lynas et al. article can itself be classed as misinformation and could therefore mislead the general public as well as the scientific community.

Background Glyphosate-based herbicides (GBHs) are the world’s most widely used weed control agents. Public health concerns have increased since the International Agency for Research on Cancer (IARC) classified glyphosate as a probable human carcinogen in 2015. To further investigate the health effects of glyphosate and GBHs, the Ramazzini Institute launched the Global Glyphosate Study (GGS), which is designed to test a wide range of toxicological outcomes. Reported here are the results of the carcinogenicity arm of the GGS. Methods Glyphosate and two GBHs, Roundup Bioflow used in the European Union (EU) and RangerPro used in the U.S., were administered to male and female Sprague–Dawley (SD) rats, beginning at gestational day 6 (via maternal exposure) through 104 weeks of age. Glyphosate was administered through drinking water at three doses: the EU acceptable daily intake (ADI) of 0.5 mg/kg body weight/day, 5 mg/kg body weight/day and the EU no-observed adverse effect level (NOAEL) of 50 mg/kg body weight/day. The two GBH formulations were administered at the same glyphosate-equivalent doses. Results In all 3 treatment groups, statistically significant dose-related increased trends or increased incidences of benign and malignant tumors at multiple anatomic sites were observed compared to historical and concurrent controls. These tumors arose in haemolymphoreticular tissues (leukemia), skin, liver, thyroid, nervous system, ovary, mammary gland, adrenal glands, kidney, urinary bladder, bone, endocrine pancreas, uterus and spleen (hemangiosarcoma). Increased incidences occurred in both sexes. Most of these involved tumors that are rare in SD rats (background incidence < 1%) with 40% of leukemias deaths in the treated groups occurring before 52 weeks of age and increased early deaths were also observed for other solid tumors. Conclusions Glyphosate and GBHs at exposure levels corresponding to the EU ADI and the EU NOAEL caused dose-related increases in incidence of multiple benign and malignant tumors in SD rats of both sexes. Early-life onset and mortality were observed for multiple tumors. These results provide robust evidence supporting IARC’s conclusion that there is “sufficient evidence of carcinogenicity [of glyphosate] in experimental animals”. Furthermore, our data are consistent with epidemiological evidence on the carcinogenicity of glyphosate and GBHs.

Background Dietary habits have a profound influence on the metabolic activity of gut microorganisms and their influence on health. Concerns have been raised as to whether the consumption of foodstuffs contaminated with pesticides can contribute to the development of chronic disease by affecting the gut microbiome. We performed the first pesticide biomonitoring survey of the British population, and subsequently used the results to perform the first pesticide association study on gut microbiome composition and function from the TwinsUK registry. Methods Dietary exposure of 186 common insecticide, herbicide, or fungicide residues and the faecal microbiome in 65 twin pairs in the UK was investigated. We evaluated if dietary habits, geographic location, or the rural/urban environment, are associated with the excretion of pesticide residues. The composition and metabolic activity of faecal microbiota was evaluated using shotgun metagenomics and metabolomics respectively. We performed a targeted urine metabolomics analysis in order to evaluate whether pesticide urinary excretion was also associated with physiological changes. Results Pyrethroid and/or organophosphorus insecticide residues were found in all urine samples, while the herbicide glyphosate was found in 53% of individuals. Food frequency questionnaires showed that residues from organophosphates were higher with increased consumption of fruit and vegetables. A total of 34 associations between pesticide residue concentrations and faecal metabolite concentrations were detected. Glyphosate excretion was positively associated with an overall increased bacterial species richness, as well as to fatty acid metabolites and phosphate levels. The insecticide metabolite Br2CA, reflecting deltamethrin exposure, was positively associated with the phytoestrogens enterodiol and enterolactone, and negatively associated with some N-methyl amino acids. Urine metabolomics performed on a subset of samples did not reveal associations with the excretion of pesticide residues. Conclusions The consumption of conventionally grown fruit and vegetables leads to higher ingestion of pesticides with unknown long-term health consequences. Our results highlight the need for future dietary intervention studies to understand effects of pesticide exposure on the gut microbiome and possible health consequences.