Discovery of bioactive microbial gene products in inflammatory bowel disease.

Microbial communities and their associated bioactive compounds1-3 are often disrupted in conditions such as the inflammatory bowel diseases (IBD)4. However, even in well-characterized environments (for example, the human gastrointestinal tract), more than one-third of microbial proteins are uncharacterized and often expected to be bioactive5-7. Here we systematically identified more than 340,000 protein families as potentially bioactive with respect to gut inflammation during IBD, about half of which have not to our knowledge been functionally characterized previously on the basis of homology or experiment. To validate prioritized microbial proteins, we used a combination of metagenomics, metatranscriptomics and metaproteomics to provide evidence of bioactivity for a subset of proteins that are involved in host and microbial cell-cell communication in the microbiome; for example, proteins associated with adherence or invasion processes, and extracellular von Willebrand-like factors. Predictions from high-throughput data were validated using targeted experiments that revealed the differential immunogenicity of prioritized Enterobacteriaceae pilins and the contribution of homologues of von Willebrand factors to the formation of Bacteroides biofilms in a manner dependent on mucin levels. This methodology, which we term MetaWIBELE (workflow to identify novel bioactive elements in the microbiome), is generalizable to other environmental communities and human phenotypes. The prioritized results provide thousands of candidate microbial proteins that are likely to interact with the host immune system in IBD, thus expanding our understanding of potentially bioactive gene products in chronic disease states and offering a rational compendium of possible therapeutic compounds and targets.

Statistical approaches for differential expression analysis in metatranscriptomics.

MOTIVATION: Metatranscriptomics (MTX) has become an increasingly practical way to profile the functional activity of microbial communities in situ. However, MTX remains underutilized due to experimental and computational limitations. The latter are complicated by non-independent changes in both RNA transcript levels and their underlying genomic DNA copies (as microbes simultaneously change their overall abundance in the population and regulate individual transcripts), genetic plasticity (as whole loci are frequently gained and lost in microbial lineages) and measurement compositionality and zero-inflation. Here, we present a systematic evaluation of and recommendations for differential expression (DE) analysis in MTX. RESULTS: We designed and assessed six statistical models for DE discovery in MTX that incorporate different combinations of DNA and RNA normalization and assumptions about the underlying changes of gene copies or species abundance within communities. We evaluated these models on multiple simulated and real multi-omic datasets. Models adjusting transcripts relative to their encoding gene copies as a covariate were significantly more accurate in identifying DE from MTX in both simulated and real datasets. Moreover, we show that when paired DNA measurements (metagenomic data) are not available, models normalizing MTX measurements within-species while also adjusting for total-species RNA balance sensitivity, specificity and interpretability of DE detection, as does filtering likely technical zeros. The efficiency and accuracy of these models pave the way for more effective MTX-based DE discovery in microbial communities. AVAILABILITY AND IMPLEMENTATION: The analysis code and synthetic datasets used in this evaluation are available online at http://huttenhower.sph.harvard.edu/mtx2021. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Multivariable association discovery in population-scale meta-omics studies.

It is challenging to associate features such as human health outcomes, diet, environmental conditions, or other metadata to microbial community measurements, due in part to their quantitative properties. Microbiome multi-omics are typically noisy, sparse (zero-inflated), high-dimensional, extremely non-normal, and often in the form of count or compositional measurements. Here we introduce an optimized combination of novel and established methodology to assess multivariable association of microbial community features with complex metadata in population-scale observational studies. Our approach, MaAsLin 2 (Microbiome Multivariable Associations with Linear Models), uses generalized linear and mixed models to accommodate a wide variety of modern epidemiological studies, including cross-sectional and longitudinal designs, as well as a variety of data types (e.g., counts and relative abundances) with or without covariates and repeated measurements. To construct this method, we conducted a large-scale evaluation of a broad range of scenarios under which straightforward identification of meta-omics associations can be challenging. These simulation studies reveal that MaAsLin 2's linear model preserves statistical power in the presence of repeated measures and multiple covariates, while accounting for the nuances of meta-omics features and controlling false discovery. We also applied MaAsLin 2 to a microbial multi-omics dataset from the Integrative Human Microbiome (HMP2) project which, in addition to reproducing established results, revealed a unique, integrated landscape of inflammatory bowel diseases (IBD) across multiple time points and omics profiles.

Effects of Lifetime Exposures to Environmental Contaminants on the Adult Gut Microbiome.

Emerging experimental evidence indicates that toxicant-induced alterations in gut microbiota composition and activity may affect host homeostasis. However, data from human studies are scarce; to our knowledge, no previous studies have quantified the association of lifetime exposure to environmental chemicals, across multiple time points, with the composition of the adult gut microbiome. Here we studied 124 individuals born in the Faroe Islands in 1986-1987 who were followed approximately every seven years from birth through age 28 years. Organochlorine compounds, including polychlorinated biphenyls (PCBs) and pesticides, perfluoroalkyl substances (PFAS), and mercury (Hg), were measured in cord blood and longitudinally in participants' blood. At age 28, the gut microbiome was assessed using shotgun metagenomic sequencing. Historical contaminant exposures had little direct effect on the adult gut microbiome, while a small number of fastidious anaerobes were weakly linked to recent PFAS/PFOS exposures at age 28. In this cohort, our findings suggest no lasting effects of early life exposures on adult gut microbial composition, but proximal exposures may contribute to gut microbiome alterations. The methods developed and used for this investigation may help in future identification of small but lasting impacts of environmental toxicant exposure on the gut microbiome.

Trophic-Level Interactive Effects of Phosphorus Availability on the Toxicities of Cadmium, Arsenic, and Their Binary Mixture in Media-Exposed Scenedesmus acutus and Media and Dietary-Exposed Daphnia pulex.

Various anthropogenic activities simultaneously alter essential mineral nutrients and contaminant content in the environment. Depending on essential nutrient conditions, the uptake and effects of contaminants in exposed organisms may be altered. The addressing of ecological risk assessment (ERA) of contaminant mixtures has proven difficult. Furthermore, most assessments involving single contaminant exposures do not consider the interaction of essential nutrients on toxicological end points. Hypotheses for toxicological effects of cadmium (Cd), arsenic (As), and their binary mixture (Cd/Asmix) include alteration under varying dietary and media phosphorus (P) conditions. However, interactive effects and effect size (η2) are largely unknown. Here, we investigated the toxicities of Cd-, As-, and Cd/Asmix-treated media and diets on Scenedesmus acutus (a primary producer) and Daphnia pulex (a primary consumer), under varied media and dietary P conditions [low (LP), median (MP), and optimum (COMBO)]. Our results showed significant (p < 0.05) interactive effects and concentration dependent growth inhibition of S. acutus. The toxicity (at day 7) of Cd against S. acutus was 2×, 11×, and 4× that of As in LP, MP, and COMBO conditions, respectively, while the joint toxicity effects of Cd/Asmix were partially additive in LP and COMBO, and synergistic in MP media. Furthermore, acute lethal toxicity (96 h) of Cd in D. pulex was ∼60× that of As, while Cd/Asmix joint toxicity was synergistic. Chronic toxicity (14 d) in D. pulex showed significant (p < 0.05) interaction of As and P-availability on survival, reproduction, and behavior (distance moved, velocity, acceleration and mobility), while Cd and P availability showed significant interactive effect on rotational behavior. Dose response effects of Cd, As, and Cd/Asmix in S. acutus and D. pulex were either monophasic or biphasic under varying nutrient conditions. This study provides empirical evidence of the interactive effects of media/dietary P and toxic metals (Cd, As, and Cd/Asmix) at environmentally relevant concentrations, emphasizing the need for consideration of such interactions during ERA.

RNA-based amplicon sequencing is ineffective in measuring metabolic activity in environmental microbial communities.

BACKGROUND: Characterization of microbial activity is essential to the understanding of the basic biology of microbial communities, as the function of a microbiome is defined by its biochemically active ("viable") community members. Current sequence-based technologies can rarely differentiate microbial activity, due to their inability to distinguish live and dead sourced DNA. As a result, our understanding of microbial community structures and the potential mechanisms of transmission between humans and our surrounding environments remains incomplete. As a potential solution, 16S rRNA transcript-based amplicon sequencing (16S-RNA-seq) has been proposed as a reliable methodology to characterize the active components of a microbiome, but its efficacy has not been evaluated systematically. Here, we present our work to benchmark RNA-based amplicon sequencing for activity assessment in synthetic and environmentally sourced microbial communities. RESULTS: In synthetic mixtures of living and heat-killed Escherichia coli and Streptococcus sanguinis, 16S-RNA-seq successfully reconstructed the active compositions of the communities. However, in the realistic environmental samples, no significant compositional differences were observed in RNA ("actively transcribed - active") vs. DNA ("whole" communities) spiked with E. coli controls, suggesting that this methodology is not appropriate for activity assessment in complex communities. The results were slightly different when validated in environmental samples of similar origins (i.e., from Boston subway systems), where samples were differentiated both by environment type as well as by library type, though compositional dissimilarities between DNA and RNA samples remained low (Bray-Curtis distance median: 0.34-0.49). To improve the interpretation of 16S-RNA-seq results, we compared our results with previous studies and found that 16S-RNA-seq suggests taxon-wise viability trends (i.e., specific taxa are universally more or less likely to be viable compared to others) in samples of similar origins. CONCLUSIONS: This study provides a comprehensive evaluation of 16S-RNA-seq for viability assessment in synthetic and complex microbial communities. The results found that while 16S-RNA-seq was able to semi-quantify microbial viability in relatively simple communities, it only suggests a taxon-dependent "relative" viability in realistic communities.  Video Abstract.

Extending and improving metagenomic taxonomic profiling with uncharacterized species using MetaPhlAn 4.

Metagenomic assembly enables new organism discovery from microbial communities, but it can only capture few abundant organisms from most metagenomes. Here we present MetaPhlAn 4, which integrates information from metagenome assemblies and microbial isolate genomes for more comprehensive metagenomic taxonomic profiling. From a curated collection of 1.01 M prokaryotic reference and metagenome-assembled genomes, we define unique marker genes for 26,970 species-level genome bins, 4,992 of them taxonomically unidentified at the species level. MetaPhlAn 4 explains ~20% more reads in most international human gut microbiomes and >40% in less-characterized environments such as the rumen microbiome and proves more accurate than available alternatives on synthetic evaluations while also reliably quantifying organisms with no cultured isolates. Application of the method to >24,500 metagenomes highlights previously undetected species to be strong biomarkers for host conditions and lifestyles in human and mouse microbiomes and shows that even previously uncharacterized species can be genetically profiled at the resolution of single microbial strains.

Metagenomic assessment of gut microbial communities and risk of severe COVID-19.

BACKGROUND: The gut microbiome is a critical modulator of host immunity and is linked to the immune response to respiratory viral infections. However, few studies have gone beyond describing broad compositional alterations in severe COVID-19, defined as acute respiratory or other organ failure. METHODS: We profiled 127 hospitalized patients with COVID-19 (n = 79 with severe COVID-19 and 48 with moderate) who collectively provided 241 stool samples from April 2020 to May 2021 to identify links between COVID-19 severity and gut microbial taxa, their biochemical pathways, and stool metabolites. RESULTS: Forty-eight species were associated with severe disease after accounting for antibiotic use, age, sex, and various comorbidities. These included significant in-hospital depletions of Fusicatenibacter saccharivorans and Roseburia hominis, each previously linked to post-acute COVID syndrome or "long COVID," suggesting these microbes may serve as early biomarkers for the eventual development of long COVID. A random forest classifier achieved excellent performance when tasked with classifying whether stool was obtained from patients with severe vs. moderate COVID-19, a finding that was externally validated in an independent cohort. Dedicated network analyses demonstrated fragile microbial ecology in severe disease, characterized by fracturing of clusters and reduced negative selection. We also observed shifts in predicted stool metabolite pools, implicating perturbed bile acid metabolism in severe disease. CONCLUSIONS: Here, we show that the gut microbiome differentiates individuals with a more severe disease course after infection with COVID-19 and offer several tractable and biologically plausible mechanisms through which gut microbial communities may influence COVID-19 disease course. Further studies are needed to expand upon these observations to better leverage the gut microbiome as a potential biomarker for disease severity and as a target for therapeutic intervention.

Alterations in the gut microbiome implicate key taxa and metabolic pathways across inflammatory arthritis phenotypes.

Musculoskeletal diseases affect up to 20% of adults worldwide. The gut microbiome has been implicated in inflammatory conditions, but large-scale metagenomic evaluations have not yet traced the routes by which immunity in the gut affects inflammatory arthritis. To characterize the community structure and associated functional processes driving gut microbial involvement in arthritis, the Inflammatory Arthritis Microbiome Consortium investigated 440 stool shotgun metagenomes comprising 221 adults diagnosed with rheumatoid arthritis, ankylosing spondylitis, or psoriatic arthritis and 219 healthy controls and individuals with joint pain without an underlying inflammatory cause. Diagnosis explained about 2% of gut taxonomic variability, which is comparable in magnitude to inflammatory bowel disease. We identified several candidate microbes with differential carriage patterns in patients with elevated blood markers for inflammation. Our results confirm and extend previous findings of increased carriage of typically oral and inflammatory taxa and decreased abundance and prevalence of typical gut clades, indicating that distal inflammatory conditions, as well as local conditions, correspond to alterations to the gut microbial composition. We identified several differentially encoded pathways in the gut microbiome of patients with inflammatory arthritis, including changes in vitamin B salvage and biosynthesis and enrichment of iron sequestration. Although several of these changes characteristic of inflammation could have causal roles, we hypothesize that they are mainly positive feedback responses to changes in host physiology and immune homeostasis. By connecting taxonomic alternations to functional alterations, this work expands our understanding of the shifts in the gut ecosystem that occur in response to systemic inflammation during arthritis.

Metatranscriptomics for the Human Microbiome and Microbial Community Functional Profiling.

Shotgun metatranscriptomics (MTX) is an increasingly practical way to survey microbial community gene function and regulation at scale. This review begins by summarizing the motivations for community transcriptomics and the history of the field. We then explore the principles, best practices, and challenges of contemporary MTX workflows: beginning with laboratory methods for isolation and sequencing of community RNA, followed by informatics methods for quantifying RNA features, and finally statistical methods for detecting differential expression in a community context. In thesecond half of the review, we survey important biological findings from the MTX literature, drawing examples from the human microbiome, other (nonhuman) host-associated microbiomes, and the environment. Across these examples, MTX methods prove invaluable for probing microbe-microbe and host-microbe interactions, the dynamics of energy harvest and chemical cycling, and responses to environmental stresses. We conclude with a review of open challenges in the MTX field, including making assays and analyses more robust, accessible, and adaptable to new technologies; deciphering roles for millions of uncharacterized microbial transcripts; and solving applied problems such as biomarker discovery and development of microbial therapeutics.

Use of nest bundles to monitor agrochemical exposure and effects among cavity nesting pollinators.

Cavity nesting bees are proficient and important pollinators that can augment or replace honey bee pollination services for some crops. Relatively little is known about specific pesticide concentrations present in cavity nesting insect reed matrices and associated potential risks to cavity nesting bees. Nesting substrates (Phragmites australis reeds in bundles) were deployed in an agriculturally intensive landscape to evaluate colonization and agrochemical exposure among cavity nesting pollinators over two consecutive field seasons. Composition of insect species colonizing reeds within nest bundles varied considerably; those placed near beef cattle feed yards were dominated by wasps (93% of the total number of individuals occupying reed nest bundles), whereas nest bundles deployed in cropland-dominated landscapes were colonized primarily by leaf cutter bees (71%). All nesting/brood matrices in reeds (mud, leaves, brood, pollen) contained agrochemicals. Mud used in brood chamber construction at feed yard sites contained 21 of 23 agrochemicals included in analysis and >70% of leaf substrate stored in reeds contained at least one agrochemical. Moxidectin was most frequently detected across all reed matrices from feed yard sites, and moxidectin concentrations in nonviable larvae were more than four times higher than those quantified in viable larvae. Agrochemical concentrations in leaf material and pollen were also quantified at levels that may have induced toxic effects among developing larvae. To our knowledge, this is the first study to characterize agrochemical concentrations in multiple reed matrices provisioned by cavity-nesting insects. Use of nest bundles revealed that cavity nesting pollinators in agriculturally intensive regions are exposed to agrochemicals during all life stages, at relatively high frequencies, and at potentially lethal concentrations. These results demonstrate the utility of nest bundles for characterizing risks to cavity nesting insects inhabiting agriculturally intensive regions.

Whole microbial community viability is not quantitatively reflected by propidium monoazide sequencing approach.

BACKGROUND: High-throughput sequencing provides a powerful window into the structural and functional profiling of microbial communities, but it is unable to characterize only the viable portion of microbial communities at scale. There is as yet not one best solution to this problem. Previous studies have established viability assessments using propidium monoazide (PMA) treatment coupled with downstream molecular profiling (e.g., qPCR or sequencing). While these studies have met with moderate success, most of them focused on the resulting "viable" communities without systematic evaluations of the technique. Here, we present our work to rigorously benchmark "PMA-seq" (PMA treatment followed by 16S rRNA gene amplicon sequencing) for viability assessment in synthetic and realistic microbial communities. RESULTS: PMA-seq was able to successfully reconstruct simple synthetic communities comprising viable/heat-killed Escherichia coli and Streptococcus sanguinis. However, in realistically complex communities (computer screens, computer mice, soil, and human saliva) with E. coli spike-in controls, PMA-seq did not accurately quantify viability (even relative to variability in amplicon sequencing), with its performance largely affected by community properties such as initial biomass, sample types, and compositional diversity. We then applied this technique to environmental swabs from the Boston subway system. Several taxa differed significantly after PMA treatment, while not all microorganisms responded consistently. To elucidate the "PMA-responsive" microbes, we compared our results with previous PMA-based studies and found that PMA responsiveness varied widely when microbes were sourced from different ecosystems but were reproducible within similar environments across studies. CONCLUSIONS: This study provides a comprehensive evaluation of PMA-seq exploring its quantitative potential in synthetic and complex microbial communities, where the technique was effective for semi-quantitative purposes in simple synthetic communities but provided only qualitative assessments in realistically complex community samples. Video abstract.

Microbiome Analysis of More Than 2,000 NHS Bowel Cancer Screening Programme Samples Shows the Potential to Improve Screening Accuracy.

PURPOSE: There is potential for fecal microbiome profiling to improve colorectal cancer screening. This has been demonstrated by research studies, but it has not been quantified at scale using samples collected and processed routinely by a national screening program. EXPERIMENTAL DESIGN: Between 2016 and 2019, the largest of the NHS Bowel Cancer Screening Programme hubs prospectively collected processed guaiac fecal occult blood test (gFOBT) samples with subsequent colonoscopy outcomes: blood-negative [n = 491 (22%)]; colorectal cancer [n = 430 (19%)]; adenoma [n = 665 (30%)]; colonoscopy-normal [n = 300 (13%)]; nonneoplastic [n = 366 (16%)]. Samples were transported and stored at room temperature. DNA underwent 16S rRNA gene V4 amplicon sequencing. Taxonomic profiling was performed to provide features for classification via random forests (RF). RESULTS: Samples provided 16S amplicon-based microbial profiles, which confirmed previously described colorectal cancer-microbiome associations. Microbiome-based RF models showed potential as a first-tier screen, distinguishing colorectal cancer or neoplasm (colorectal cancer or adenoma) from blood-negative with AUC 0.86 (0.82-0.89) and AUC 0.78 (0.74-0.82), respectively. Microbiome-based models also showed potential as a second-tier screen, distinguishing from among gFOBT blood-positive samples, colorectal cancer or neoplasm from colonoscopy-normal with AUC 0.79 (0.74-0.83) and AUC 0.73 (0.68-0.77), respectively. Models remained robust when restricted to 15 taxa, and performed similarly during external validation with metagenomic datasets. CONCLUSIONS: Microbiome features can be assessed using gFOBT samples collected and processed routinely by a national colorectal cancer screening program to improve accuracy as a first- or second-tier screen. The models required as few as 15 taxa, raising the potential of an inexpensive qPCR test. This could reduce the number of colonoscopies in countries that use fecal occult blood test screening.

The colorectal cancer-associated faecal microbiome of developing countries resembles that of developed countries.

BACKGROUND: The incidence of colorectal cancer (CRC) is increasing in developing countries, yet limited research on the CRC- associated microbiota has been conducted in these areas, in part due to scarce resources, facilities, and the difficulty of fresh or frozen stool storage/transport. Here, we aimed (1) to establish a broad representation of diverse developing countries (Argentina, Chile, India, and Vietnam); (2) to validate a 'resource-light' sample-collection protocol translatable in these settings using guaiac faecal occult blood test (gFOBT) cards stored and, importantly, shipped internationally at room temperature; (3) to perform initial profiling of the collective CRC-associated microbiome of these developing countries; and (4) to compare this quantitatively with established CRC biomarkers from developed countries. METHODS: We assessed the effect of international storage and transport at room temperature by replicating gFOBT from five UK volunteers, storing two in the UK, and sending replicates to institutes in the four countries. Next, to determine the effect of prolonged UK storage, DNA extraction replicates for a subset of samples were performed up to 252 days apart. To profile the CRC-associated microbiome of developing countries, gFOBT were collected from 41 treatment-naïve CRC patients and 40 non-CRC controls from across the four institutes, and V4 16S rRNA gene sequencing was performed. Finally, we constructed a random forest (RF) model that was trained and tested against existing datasets from developed countries. RESULTS: The microbiome was stably assayed when samples were stored/transported at room temperature and after prolonged UK storage. Large-scale microbiome structure was separated by country and continent, with a smaller effect from CRC. Importantly, the RF model performed similarly to models trained using external datasets and identified similar taxa of importance (Parvimonas, Peptostreptococcus, Fusobacterium, Alistipes, and Escherichia). CONCLUSIONS: This study demonstrates that gFOBT, stored and transported at room temperature, represents a suitable method of faecal sample collection for amplicon-based microbiome biomarkers in developing countries and suggests a CRC-faecal microbiome association that is consistent between developed and developing countries.

The Sulfur Microbial Diet and Risk of Colorectal Cancer by Molecular Subtypes and Intratumoral Microbial Species in Adult Men.

INTRODUCTION: We recently described the sulfur microbial diet, a pattern of intake associated with increased gut sulfur-metabolizing bacteria and incidence of distal colorectal cancer (CRC). We assessed whether this risk differed by CRC molecular subtypes or presence of intratumoral microbes involved in CRC pathogenesis (Fusobacterium nucleatum and Bifidobacterium spp.). METHODS: We performed Cox proportional hazards modeling to examine the association between the sulfur microbial diet and incidence of overall and distal CRC by molecular and microbial subtype in the Health Professionals Follow-Up Study (1986-2012). RESULTS: We documented 1,264 incident CRC cases among 48,246 men, approximately 40% of whom had available tissue data. After accounting for multiple hypothesis testing, the relationship between the sulfur microbial diet and CRC incidence did not differ by subtype. However, there was a suggestion of an association by prostaglandin synthase 2 (PTGS2) status with a multivariable adjusted hazard ratio for highest vs lowest tertile of sulfur microbial diet scores of 1.31 (95% confidence interval: 0.99-1.74, Ptrend = 0.07, Pheterogeneity = 0.04) for PTGS2-high CRC. The association of the sulfur microbial diet with distal CRC seemed to differ by the presence of intratumoral Bifidobacterium spp. with an adjusted hazard ratio for highest vs lowest tertile of sulfur microbial diet scores of 1.65 (95% confidence interval: 1.14-2.39, Ptrend = 0.01, Pheterogeneity = 0.03) for Bifidobacterium-negative distal CRC. We observed no apparent heterogeneity by other tested molecular markers. DISCUSSION: Greater long-term adherence to the sulfur microbial diet could be associated with PTGS2-high and Bifidobacterium-negative distal CRC in men. Additional studies are needed to further characterize the role of gut microbial sulfur metabolism and CRC.

Nitrate-N-mediated toxicological responses of Scenedesmus acutus and Daphnia pulex to cadmium, arsenic and their binary mixture (Cd/Asmix) at environmentally relevant concentrations.

Several biomarkers used for ecological risk assessment have been established for single contaminant toxicity, many of which are less predictive of the influence of media and/or dietary nutrients on toxicity outcomes of contaminant mixtures. In this study, we investigate toxicological responses and life traits of Scenedesmus acutus and Daphnia pulex to heavy metals (cadmium-Cd, arsenic-As, binary mixture-Cd/Asmix) in media and diets with varied nutrient (nitrate-N) conditions (low-LN, median-MN, optimum-COMBO). Results showed that nitrate-N-mediated metal inhibitory effects on growth and productivity of primary producer (S. acutus) were significantly interactive (p < 0.05; effect size, ƞ2≤56 %). Cadmium toxicities (Cd-IC50s) in S. acutus were 1.2×, 5.3×, and 4.3× As-IC50s in LN, MN and COMBO media, respectively, while mixture (Cd/Asmix) toxicities were synergistic in MN medium and partial additivity in COMBO and LN media. Nitrate-N and metal exposure effects on S. acutus nutrient stoichiometry, metal uptake and bioaccumulation were significantly interactive (p < 0.05, ƞ2≤100 %). Moreover, survival of primary consumer (D. pulex) was significantly impaired by single and mixed dietary-metal exposures with greater effect under LN condition coupled with significant interactive effects on reproductive capacity (p < 0.05, ƞ2≤21.2 %) but not on swimming activity. We recommend that nitrate-N-mediated metal exposure effects/toxicity in bioindicator species should be considered during ecological risk assessments.

Homologous recombination repair signaling in chemical carcinogenesis: prolonged particulate hexavalent chromium exposure suppresses the Rad51 response in human lung cells.

The aim of this study was to focus on hexavalent chromium, [Cr(VI)], a chemical carcinogen and major public health concern, and consider its ability to impact DNA double strand break repair. We further focused on particulate Cr(VI), because it is the more potent carcinogenic form of Cr(VI). DNA double strand break repair serves to protect cells against the detrimental effects of DNA double strand breaks. For particulate Cr(VI), data show DNA double strand break repair must be overcome for neoplastic transformation to occur. Acute Cr(VI) exposures reveal a robust DNA double strand break repair response, however, longer exposures have not been considered. Using the comet assay, we found longer exposures to particulate zinc chromate induced concentration-dependent increases in DNA double strand breaks indicating breaks were occurring throughout the exposure time. Acute (24 h) exposure induced DNA double strand break repair signaling by inducing Mre11 foci formation, ATM phosphorylation and phosphorylated ATM foci formation, Rad51 protein levels and Rad51 foci formation. However, longer exposures reduced the Rad51 response. These data indicate a major chemical carcinogen can simultaneously induce DNA double strand breaks and alter their repair and describe a new and important aspect of the carcinogenic mechanism for Cr(VI).