The human gut microbiota makes key contributions to the metabolism of ingested compounds (xenobiotics), transforming hundreds of dietary components, industrial chemicals, and pharmaceuticals into metabolites with altered activities, toxicities, and lifetimes within the body. The chemistry of gut microbial xenobiotic metabolism is often distinct from that of host enzymes. Despite their important consequences for human biology, the gut microbes, genes, and enzymes involved in xenobiotic metabolism are poorly understood. Linking these microbial transformations to enzymes and elucidating their biological effects is undoubtedly challenging. However, recent studies demonstrate that integrating traditional and emerging technologies can enable progress toward this goal. Ultimately, a molecular understanding of gut microbial xenobiotic metabolism will guide personalized medicine and nutrition, inform toxicology risk assessment, and improve drug discovery and development.Humans ingest a multitude of smallmolecules that are foreign to the body (xenobiotics), including dietary components, environmental chemicals, and pharmaceuticals. The trillions of microorganisms that inhabit our gastrointestinal tract (the human gut microbiota) can directly alter the chemical structures of such compounds, thus modifying their lifetimes, bioavailabilities, and biological effects. Our knowledge of how gut microbial transformations of xenobiotics affect human health is in its infancy, which is surprising given the importance of the gut microbiota. We currently lack an understanding of the extent to which this metabolism varies between individuals, the mechanisms by which these microbial activities influence human biology, and how we might rationally manipulate these reactions. This deficiency stems largely from the difficulty of connecting this microbial chemistry to specific organisms, genes, and enzymes.Over the past several decades, studies of gut microbiota–mediated modification of xenobiotics have revealed that these organisms collectively have a larger metabolic repertoire than human cells. The chemical differences between human andmicrobial transformations of ingested compounds arise not only from the increased diversity of enzymes present in this complex and variable community but also from the distinct selection pressures that have shaped these activities. For example, whereas host metabolism evolved to facilitate excretion of many xenobiotics from the body, microbial modifications of these compounds and their human metabolites often support microbial growth through provision of nutrients or production of energy. Notably, the chemistry of microbial transformations often opposes or reverses that of host metabolism, altering the pharmacokinetic and pharmacodynamic properties of xenobiotics and associated metabolites.The range of xenobiotics subject to gutmicrobial metabolism is impressive and expanding. Gut microbes modify many classes of dietary compounds, including complex polysaccharides, lipids, proteins, and phytochemicals. These metabolic reactions are linked to a variety of health benefits, aswell as disease susceptibilities. Gut microbes are also able to transform industrial chemicals and pollutants, altering their toxicities and lifetimes in the body. Similarly, microbial transformations of drugs can change their pharmacokinetic properties, be critical for prodrug activation, and lead to undesirable side effects or loss of efficacy. In the vast majority of cases, the individual microbes and enzymes that mediate these reactions are unknown.Fueled by findings underscoring the relevance of microbial xenobiotic metabolism to human health, scientists are increasingly seeking to discover and manipulate the enzymatic chemistry involved in these transformations. Recent work exploring how gut microbes metabolize the drugs digoxin and irinotecan, as well as the dietary nutrient choline, provides guidance for such investigations. These studies, which combine traditional methods with modern approaches, illustrate how a molecular understanding of gut microbial xenobiotic metabolism can guide hypothesis-driven research into the roles these reactions play in both microbiota and host biology.We still face a myriad of challenges in understanding the gut microbiota’s contribution to xenobiotic metabolism. It is imperative that we connect the many known microbial transformations with the genes and enzymes responsible for these activities, and knowledge of enzyme mechanism and biochemical logic will facilitate this objective. There also remains a great need to uncover currently unappreciated activities associated with this community. Revealing the full scope of microbially mediated transformations in the gut may give us newinsights into themany variable and contradictory studies regarding the effects of diet, pollutants, and drugs on human health. Microbial genes and enzymes will provide both specific targets for manipulation and diagnostic markers that can be incorporated into clinical studies and practice. Ultimately, a molecular understanding of gut microbial xenobiotic metabolism will inform personalized nutrition, toxicology risk assessment, precision medicine, and drug development. The estimated 1013 microbes that inhabit the human gastrointestinal (GI) tract play a central role in many processes, including colonization resistance, immune system modulation, synthesis of essential vitamins and nutrients, and digestion of polysaccharides (2–4). Gut microbes also modify the chemical structures of numerous ingested, foreign compounds (xenobiotics), including dietary components, environmental pollutants, and pharmaceuticals. Such transformations were identified as early as the 1950s in humans, animal models, fecal samples, and individual microbes. In these studies, changes in metabolism in the absence of microbes  or upon microbial perturbation  indicated the gut microbiota’s involvement in xenobiotic processing .The human gut microbiota is a diverse and complex community of microorganisms that has evolved with its host . Hundreds of distinct microbial species colonize the human gut. Although obligate anaerobes such as the Firmicutes and Bacteroidetes phyla typically dominate, large variability in community composition is observed among individuals .The majority of human microbiota-xenobiotic interactions occur within the GI tract. The different regions of this organ system vary in epithelial cell physiology, pH, oxygen levels, and nutrient content, thus providing distinct habitats for microorganisms and influencing the types of metabolic processes that occur (11). Readily absorbed xenobiotics pass between or through intestinal epithelial cells, where they may be processed by host enzymes before transport to the liver via the portal vein. Following exposure to the liver’s rich collection of metabolic enzymes, xenobiotics and their metabolites enter systemic circulation, distributing into tissues and potentially affecting distal organs. By contrast, intravenously administered compounds circumvent this “firstpass” metabolism and are immediately introduced into systemic circulation. Compounds in the circulatory system are eventually further metabolized and/or excreted, which generally occurs either via the biliary duct back into the gut lumen (biliary excretion) or through the kidneys into the urine. Metabolites returned to the gut lumen can either continue on to the large intestine, where they will eventually be excreted in the feces, or they can potentially be reabsorbed by host cells in the small intestine through a process known as enterohepatic circulation.Microbial metabolism of xenobiotics must be understood in the context of the concurrent and often competing metabolic processes occurring in the human host. Orally ingested compounds pass through the upper GI tract to the small intestine where they can be modified by digestive enzymes and absorbed by host tissues  can also reach gut microbes through biliary excretion. The products of gut microbialmetabolism can be absorbed by the host and circulated systemically or interact locally with the epithelial cells lining the GI tract. Ultimately, these microbial metabolites are excreted in feces or filtered by the kidneys and eliminated in the urine. Overall, human and microbial transformations generate a complex intertwined metabolic network that affects both the host and the members of the microbiota.5), many of which are distinct to these organisms. This is in stark contrast to host enzymes, which typically use oxidative and conjugative chemistry. These differences are partially due to physiological context, but they also reflect distinct evolutionary pressures. Additionally, it is likely that certain gut microbial enzymes were not evolutionarily selected to process specific xenobiotics, but rather that metabolism arises from relaxed substrate specificity. Thus, the combined metabolisms of host and microbiota generate metabolites that would not be synthesized by the host alone and can substantially alter the bioactivities and lifetimes of xenobiotics within the human body.Within the distinctive and complex ecology of the human gut, microorganisms transform ingested substrates via a broad range of enzymatic reactions. Gut microbes primarily use hydrolytic and reductive reactions tometabolize xenobiotics (12–16). Metagenomic analyses have also revealed them to be among the most prevalent protein families in this environment . It is therefore likely thatmany important transformations of xenobiotics may be performed by multiple different phylogenetic groups of gut microbes.However, it is critical to note that broad annotations are not predictive of substrate specificity, as enzymes with high sequence similarity can catalyze distinct chemical reactions.Metabolic activities can also be discontinuously distributed across closely related strains and acquired via horizontal gene transfer, making it problematic to infer gut microbial metabolic capabilities from phylogenetic analyses alone. This issue highlights the value of culture-based experiments and rigorous biochemical characterization of gut microbial enzymes in understanding xenobiotic metabolism.Many of the enzyme classes associated with xenobiotic metabolism  and highlighted here are widely distributed among sequenced gut microorganisms (11). Transferase enzymes predominate in phase IImetabolism, appending glucuronyl,methyl, acetyl, sulfonyl, and glutathionyl groups onto xenobiotics or phase Imetabolites (19). Polymorphisms in xenobiotic-metabolizing genes influence how individuals respond to both dietary and pharmaceutical interventions.Any discussion of microbial xenobiotic transformations must also consider host chemical capabilities. Human xenobioticmetabolism generally transforms nonpolar compounds into hydrophilic, higher–molecular weightmetabolites that aremore readily excreted . This prBox 1The chemistry of gut microbial and host xenobiotic metabolism. (A) Chemical logic of host xenobiotic metabolism. Commonly used chemical strategies for microbial xenobiotic metabolism include (B) hydrolytic transformations, (C) lyase reactions, (D) reductive transformations, (E) functional group transfer reactions, and (F) transformations mediated by radical enzymes. Enz, enzyme; PLP, pyridoxal 5-phosphate; NAD(P)H, NADH or NADPH; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; Me, methyl; CoA, coenzyme A; SAM, S-adenosylmethionine.20) with different substrate specificities and potentially different clinical consequences (21). Glycosidases hydrolyze glycosidic bonds using a dyad of carboxylic acid residues and a water molecule, releasing free sugars (22). These enzymes process a huge diversity of glycoconjugates and oligosaccharides and are broadly distributed across gut microbes . Sulfatases, which are also widespread, hydrolyze sulfate esters generated by phase II host metabolism using the unusual amino acid formylglycine (24). The hydrate form of this residue is thought to undergo transesterification with a sulfate ester substrate to generate a tetrahedral intermediate that breaks down to release sulfate and reform the aldehyde (25).Both the host and gut microbiota use hydrolytic chemistry to break down large ingested compounds into smaller products that may be further metabolized. Hydrolase enzymes catalyze the addition of a water molecule to a substrate, followed by bond cleavage . The mos26, 27). Hydrolysis can also alter the biological activity or toxicity of xenobiotics, as observed for plant-derived glycosides like amygdalin and the artificial sweetener cyclamate . Moreover, hydrolysis is often a prerequisite for further transformations, such as the fermentation of sugars released from indiges indigestible polysaccharides (30), and the products of hydrolytic reactions  often support microbial growth and survival in the gut.Hydrolytic reactions alter both the physical properties and activities of xenobiotics and their metabolites. For example, removal of a glucuronide in the gut lumen is generally accompanied by a decrease in polarity that can allow reabsorption by host cells and thereby extend the lifetime of a molecule within the body, as seen with glucuronide conjugates of nonsteroidal antiinflammatory drugs and the cancer therapy irinotecan (12). El Kaoutari et al. found that a single human gut microbiome encoded >5000 PLs (15), suggesting an enormous diversity of transformations that could support microbial growth.Lyase enzymes break C–C or C–X bonds  without relying on oxidation or the addition of water. Microbial polysaccharide lyases (PLs) modify polysaccharides that contain a glycosidic bond at the β position relative to a carboxylic acid  . The preS-conjugates of xenobiotics, which are formed by liver enzymes (31). Microbes can further metabolize these thiols, altering their physical properties and localization within the body. For example, gut bacterial C–S β-lyases cleave cysteine-S-conjugates of polychlorinated biphenyls to produce thiol metabolites that are further methylated and accumulate in lipophilic host tissues (32). The consequences of C–S lyase chemistry for the microbiota are not well understood. This activitymay derive from promiscuous PLP-dependent enzymes involved in “housekeeping” functions (31); however, ammonia generated by C–S β-lyases can serve as a sole nitrogen source (33), pointing to a potential role in nutrient acquisition.Microbial C–S β-lyases cleave C–S bonds found in both dietary compounds and cysteine- enzymes . These eN-oxide, azo, and sulfoxide groups  to substrates (34–36). Biochemical and structural characterization of gut microbial reductases has uncovered individual enzymes that display broad substrate scope and can transform multiple functional groups; consequently, their endogenous substrates and in vivo relevance are often unclear.Gut microbes can reduce a wide range of functional groups, including alkenes and α,β-unsaturated carboxylic acid derivatives, nitro, e groups . Reducta37–39). Notably, electron transfer to xenobiotics may enable anaerobic respiration in the human gut, where oxygen is largely unavailable to serve as a terminal electron acceptor. Although reductase enzymes are found in humans, many reductive transformations are exclusively microbial  and have not yet been linked to known enzymes or organisms.Reduction typically decreases the polarity of compounds and can alter charge, hybridization, and electrophilicity, which can affect the lifetimes and activities of metabolites in the body (S-adenosylmethionine (SAM).Whereas enzymes that remove acyl groups generally rely on hydrolysis, demethylating enzymes use cofactors capable of nucleophilic catalysis  . Installation and removal of these functional groups can affect the lifetimes and bioactivities of xenobiotics in various ways. For instance, acetylation can serve as a detoxification mechanism by decreasing polarity and facilitating excretion from microbial cells. A notable example is the N-acetylation of the anti-inflammatory compound 5-aminosalicylic acid (5-ASA) by microbial N-acetyltransferases (43), which yields a therapeutically inactive metabolite. Host demethylation of xenobiotics liberates polar groups for further conjugation and excretion from the body (44), but in microorganisms, demethylation can provide a carbon source for growth (41).Transferase enzymes move functional groups between two substrates via nucleophilic substitution reactions. The gut microbiota transfers methyl and acyl groups to or from xenobiotic scaffolds . Additio45). Many radical enzymes used in anaerobic metabolism share a common chemical logic. By using an enzyme- or cofactor-based radical species, these enzymes typically generate a substrate-based radical intermediate through single-electron transfer or homolytic bond cleavage  and skeletal rearrangements (cleavage . This in12)– dependent enzymes, and glycyl radical enzymes (GREs). These enzymes often mediate primary metabolism in anaerobic microbes and can directly or indirectly influence the fate of xenobiotics in the human body. Reactions catalyzed by gut microbial GREs include generation of trimethylamine (TMA) by choline trimethylaminelyase (CutC) (46) and decarboxylation of the tyrosine-derived metabolite p-hydroxyphenylacetate by p-hydroxyphenylacetate decarboxylase (47). This latter reaction produces p-cresol, amolecule that competes with xenobiotics for O-sulfation and detoxification by host enzymes (48).Key classes of gut microbial radical enzymes include radical SAM enzymes, cobalamin . The types and extent of these modifications vary substantially among individuals, presumably due to differences in the presence and abundance of gut microbial enzymes, and the bioactivities of the resulting metabolites range from beneficial to acutely toxic. As much attention has been focused on microbial metabolism of complex, plant-derived polysaccharides , we have chosen to highlight transformations of noncarbohydrate dietary components.Gut microbes process an enormous variety of dietary compounds to extract nutrients and energy (52). For instance, gluten-derived peptides generated by Pseudomonas aeruginosa, an opportunistic pathogen in CD patients, are prone to translocation across themouse intestine and elicit an enhanced gluten-specific immune response in comparison with peptides produced by Lactobacillus spp. from healthy individuals (53). Identification of specific proteases responsible for gut microbial gluten processing could not only enable a better understanding of CD but also inform therapeutic interventions for this disease1, including enzymatic or probiotic treatments.Dietary protein is necessary for supplying humans with essential amino acids, but the source and amount of protein can vary substantially between different diets. The gut lumen is rich in both host and microbial proteases, and studies increasingly indicate that differential microbial proteolytic activity may directly contribute to human disease. For example, the gut microbiota is associated with celiac disease (CD), a common autoimmune disorder characterized by an inflammatory response to dietary gluten found in wheat-based foods. This proline-rich protein evades complete digestion by host proteases, resulting in the generation of high–molecularweight, immunogenic peptides. The gut microbiota may affect CD by altering gluten proteolysis. Fecal suspensions from healthy individuals and CD patients process gluten proteins and immunogenic peptides differently . For example, gut bacteria can metabolize L-tryptophan into many products, including the antioxidant indole- 3-propionic acid, the neurotransmitter tryptamine, and indole, the latter of which can undergo hydroxylation and sulfation by hepatic enzymes to generate the uremic toxin indoxyl sulfate (55–57).Gut microbes can also metabolize amino acids obtained from dietary protein, including Lphenylalanine, 58, 59). One notable example involves dietary cholesterol, a major component of Western diets that is associated with increased risk of cardiovascular disease (60). While ingested cholesterol is absorbed in the small intestine and subsequently undergoes biliary excretion and enterohepatic circulation, gut microbial reduction of cholesterol generates coprostanol, which cannot be reabsorbed and is excreted. This transformation therefore effectively removes cholesterol from circulation. Coprostanol comprises up to 50% of the steroids in human feces (61), and GF mice colonized with microbes from high- and low-cholesterol–reducing patients produce distinct amounts of coprostanol (62). Animal experiments also suggest that cholesterolreducing bacteria may decrease serum cholesterol (61). Studies of the cholesterol-reducing gut bacteria Eubacterium coprostanoligenes indicate that coprostanol synthesis may involve oxidation to 5- cholesten-3-one followed by alkene isomerization to 4-cholesten-3-one, conjugate reduction, and ketone reduction (63). Identifying the enzymes responsible for these transformations and characterizing their abundance in patients may be particularly interesting, given that inhibition of cholesterol reabsorption is a clinically validated strategy for lowering cholesterol (64).Variable gut microbial metabolism of lipids and lipid-derived compounds is associated with a variety of human diseases (65) including soy isoflavones, lignans from flaxseed and sesame seeds, flavonoids like the catechins and gallate esters found in tea, and ellagic acid from nuts and berries (66–69). These molecules are processed using a range of transformations, including ring cleavage, demethylation, and dehydroxylation, which generally produce metabolites with higher oral bioavailability, increased bioactivity, and a correlation with lowered disease risk . Polyphenolmetabolism varies widelyamong individuals (67), and further research is needed to elucidate whether these microbial products can directly affect host biology or are biomarkers for disease susceptibility. From a chemical standpoint, studying polyphenolmetabolismwill also address fundamental gaps in our understanding of gut microbial enzymes.Identifying and characterizing gut microbial enzymes may also help us to better understand dietary compounds that are associated with health benefits. For example, numerous studies implicate the gut microbiota in metabolizing poorly absorbed, polyphenolic compounds from plantderived foods (70). A cyclamate hydrolyzing enzyme has been partially purified from a guinea pig–associated strain, but human gut microbial hydrolases with this activity have not been identified (29). Gut microbes can also metabolize the artificial sweeteners stevioside and xylitol using unknown enzymes . The gut microbiota acquires the ability to transform xylitol and cyclohexamate after prolonged exposure, suggesting that long-term ingestion of dietary components can select for particular microbial metabolic functions (73). Understanding the microbiota’s role in metabolizing components of processed foods could be important for assessing food safety and the long-term effects of food additives on human health.The microbiome may also interact with components of our diets that are added in the process of food manufacturing . For instance, although many artificial sweeteners are poorly metabolized by humans, studies demonstrate that they are susceptible to microbial transformation. Gut microbes convert the artificial sweetener cyclamate into cyclohexylamine via hydrolytic cleavage of its sulfamate linkage. Cyclamate was banned in the United States after studies suggested that cyclohexylamine was carcinogenic, and both this finding and the continued use of this sweetener remain controversial (f]quinoline (IQ) into the potentialmutagen 7-hydroxy IQ (74) and can hydrolyze IQ-glucuronide conjugates, prolonging the lifetime of IQ in the body (75). Gnotobiotic rats monoassociated with an Escherichia coli strain encoding a β-glucuronidase (uidA) have higher levels of unconjugated IQ and increased colonic DNA damage compared with rats colonized with an isogenic uidA mutant (75). These results appear to implicate the gut microbiota in the known link between charred meat and cancer (76).Finally, an understanding of gutmicrobial metabolic activities could provide new insights into the biological consequences of cooking practices and food preparation. The mutagenic potential of heterocyclic amines, poorly absorbed molecules produced during charring of meat and fish, can be altered by gut microbial metabolism. For example, gut microbes convert 2-amino- 3-methylimidazo[4,5-Although there is an emerging appreciation for the role of the gut microbiota in metabolizing pollutants and industrial chemicals, our knowledge of the specific transformations, strains, and enzymes involved lags far behind that of environmental microbes. However, it is clear that microbial activities can alter the toxicity and bioavailability of these compounds, as well as extend host exposure to harmful substances. When evaluating the safety of these compounds, it is thus crucial to consider the consequences of gut microbial metabolism. Here we discuss several types of chemicals that have been implicated in human disease risk and for which there is evidence that gut microbial metabolism affects toxicity.40). Despite their use for >150 years as textile dyes, food colorings, and pharmaceuticals, we have an incomplete understanding of the organisms and enzymes that process these molecules. The reductive cleavage of azo linkages yields aniline products, and this reaction can be performed by flavin- or NAD(P)Hdependent enzymes found in many eukaryotes and bacteria (13). Azoreductases have not yet been extensively characterized from many human gut bacterial strains in which this activity has been observed, and isolates can vary in their ability to reduce different dyes (77). The biological consequences of azo reduction vary depending on the substrate. For example, although microbial transformation of azo food dyes generates metabolites that are considered to be nontoxic , workers with long-term exposure to textile dyes have an increased risk of bladder cancers (79). Feeding azo textile dyes to conventional, but not GF, mice leads to the accumulation of the mutagenic bis-aniline benzidine in urine, which implicates microbial metabolism in increasing carcinogen exposure (80). Thus, the toxic effects of azo compounds may depend on both the individual dye backbone and the presence of specific metabolizing organisms.Gut microbes reductively metabolize azo compounds, some of the first industrially important synthetic chemicals . Subsequent studies in mice revealed that gutmicrobes deaminate melamine to generate ammonia and cyanuric acid (82), the latter of which forms an insoluble complex with melamine in vivo, leading to renal toxicity (83). Klebsiella species are associated with cyanuric acid production in mice and generate this metabolite in vitro (82), but it remains unclear whether the gut microbiota or this organism contribute to melamine toxicity in humans. As environmental bacteria use similar hydrolytic chemistry to metabolize other industrially relevant s-triazines, including the herbicide atrazine, these studies also raise the possibility that the gut microbiota may transform additional compounds in this class.Gut microbes also metabolize the 84). Depletion of the gut flora in rats and mice can result in the accumulation of methylmercury, thereby causing neurological symptoms (85). The enzymes responsible for this protective activity could include homologs of the demethylating, organomercuric lyase (MerB) and mercuric reductase (MerA), which have been identified in human isolates (86). However, the abundance of mer genes did not correlate with fecal methylmercury levels in a recent clinical study, raising the possibility of additional enzymes or indirect effects (87). Notably, incubation of a mixture of 16 metal(oid)s with suspensions from an in vitro simulator of the GI tract resulted in volatilization of manymetal species and the production of As/S compounds not previously observed in biological systems (88). These studies indicate that there aremajor gaps in our knowledge of the gut microbiota’s interactions with heavy metals and the resulting toxicological implications.In addition to organic pollutants, human gut microbes modify the structures and alter the toxicities of various heavy metals, including bismuth, arsenic, and mercury. Mercury bioaccumulates in living organisms, posing a threat to human health, and gut microbial metabolism may affect mercury toxicity and lifetime in the body. Rat fecal samples reduce methylmercury (CH3Hg+) to the less toxic inorganic mercury, thereby facilitating mercury excretion from the host . In some cases, the teratogenic (90), toxic , and even lethal (91) effects of these microbial metabolic activities were not recognized until drugs were on the market. Ongoing studies have illuminated a complex interplay between drugs and gut microbes , but we focus here on examples of direct microbial modifications.Apart from antibiotics, the human gut microbiota is known to transform >50 pharmaceuticals, spanning many indications and host targets, into metabolites with altered pharmacological properties (38). Gutmicrobes reduce sulfasalazine into sulfapyridine and the active antiinflammatory agent 5-ASA, and various intestinal bacteria can further metabolize 5-ASA into N-acetyl 5-ASA, a metabolite that lacks anti-inflammatory activity. Considerable variability in acetylation rates has been observed in human fecal samples (94). Together with differences in azo reduction, this observation could potentially explain variable therapeutic efficacy of sulfasalazine in patients. N-acetyl ASA inhibits the growth of anaerobes, including Clostridium difficile (43), which suggests that this activity could affect gut microbiome composition. This observation is particularly noteworthy given our increasing appreciation of the participation of the gut microbiota in IBD pathogenesis. Other gut microbial activities that are responsible for prodrug activation include reduction of the sulfoxide found in the anti-inflammatory compound sulindac (95) and reduction of the N-oxide of the anti-diarrheal drug loperamide (39). Gaining a better understanding of the specific organisms and enzymes responsible for these activities and their presence in patients could aid in drug selection and dosing.Multipledrugs that target theGI tract are affected by gut microbes, either by direct chemical modification or indirectly through the many interactions these organisms have with host cells in this environment. Notably, many of these agents rely on microbial metabolism for converting inactive precursors (prodrugs) to pharmaceutically active compounds. Prominent examples are antiinflammatory drugs that contain azo linkages, including the inflammatory bowel disease (IBD) medication sulfasalazine (96). Co-incubation with either E. coli or Listeria welshimeri either increased or decreased the efficacy of half of a panel of 30 anticancer drugs toward cancer cell lines. Assays with E. coli and a subset of these drugs  revealed evidence for direct chemical modification by the bacteria. Finally, the presence of E. coli altered the efficacy of chemotherapy in vivo in a manner consistent with the observed metabolic activities. These preliminary observations suggest that structural modification of drugs by gut or tumor-associated microbes could contribute to interindividual variation in cancer therapy, but examination of larger panels of microbes and detailed characterization of drug metabolites are needed to extend these findings.Patient response to chemotherapy can differ dramatically between individuals, in terms of efficacy as well as the severity of side effects, and emerging studies suggest that differences in the gut microbiota may contribute to this phenomenon. In addition to modulating the host immune system, gut microbes can directly alter the structures of cancer therapies and their metabolites, affecting their interactions with host cells. Recent work indicates that microbes may have a high potential formodifying the chemical structures of commonly used chemotherapeutics (L-dopa) is used to treat Parkinson’s disease, a condition characterized by dopaminergic-neuronal death. L-dopa crosses the blood-brain barrier,where it is decarboxylated by host enzymes to restore dopamine levels (97). However, extensive metabolism within the gut by both host and microbial enzymes affects the concentration of drug reaching the brain. Microbial decarboxylation (98) and p-dehydroxylation convert L-dopa to mtyramine, which can be further oxidized to mhydroxyphenylacetic acid (99). Differences in these activities may contribute to the substantial variation observed in patient response to L-dopa (100). Although a tyrosine decarboxylase from a foodassociated strain of Lactobacillus brevis accepts L-dopa in vitro (101), the human gut microbes and enzymes responsible for L-dopa metabolism are unknown. As studies continue to reveal connections between the gut microbiota and various neurological diseases (102), it will become increasingly important to identify and characterize additional microbial interactions with CNStargeted drugs.In addition to affecting drugs that act locally, gut microbial metabolism can also influence the efficacy of therapeutics that target distant organ systems. Many prominent examples can be found among CNS drugs. For example, oral levodopa (103), although clinical trials demonstrated no improvements in cancer survival or symptoms (104). In fact, subsequent animal experiments showed that gut microbes hydrolyze the glycosidic linkage of amygdalin to release mandelonitrile, which spontaneously breaks down to produce benzaldehyde and toxic cyanide . Gut microbes also metabolize the plant-derived benzoisoquinoline alkaloid berberine in a way that allows intestinal absorption (106) and convert ginsenosides, themajor bioactive components of ginseng, into metabolites that inhibit cytochrome P-450s and affect host xenobiotic metabolism (107). Because these remedies are not regulated to the same extent as pharmaceuticals, their modes of action are typically less well characterized. Elucidating how gut microbes process these compounds may contribute extensively to our fundamental understanding of their effects.Similar to dietary phytochemicals, gut microbes can also transform poorly absorbed constituents of herbal and traditional remedies, which can lead to potential health benefits or harmful side effects. There are many examples of the variable efficacy of traditional medicines, which may be partly due to the complexity of active ingredients, as well as to differences in gut microbial metabolism of these treatments. For example, amygdalin is amandelonitrile glycoside found in almonds and fruit pits that was used as an alternative cancer treatment in the 1960s (108), which limits the information gained from assessing the composition of the gut microbiota alone. Large-scale metagenomic analyses that claim to detect differences in putative xenobioticmetabolism pathways  have not provided experimental evidence supporting predicted changes in activities. To elucidate how gut microbial metabolism influences human health, it is critical to connect functions of interest with genes and enzymes. Recent studies show how the combination of traditional methods with emerging technologies can enable functional analysis. These examples also show how a molecular understanding of gut microbial xenobiotic metabolism can aid in manipulating these activities to benefit health outcomes.Attempts to decipher the biological consequences of gutmicrobial xenobiotic metabolism have been hindered by our poor understanding of these transformations. Although many associations exist between ingestion of xenobiotics processed by gut microbes and health status, we have limited information about the distribution of these activities in patient populations.Metabolic functions rarely correlate directly withmicrobial phylogeny, and considerable strain-level variation exists even within the same species  plant extracts were first used to treat “dropsy” (congestive heart failure) more than 230 years ago (111). The active constituent of foxglove is the cardiac glycoside digoxin, which inhibits Na+/K+ ATPases in cardiac myocytes, causing an influx of calcium and enhanced muscular contraction. Digoxin has a very narrow therapeutic window, requiring careful monitoring to avoid toxicity. More than 10% of patients taking digoxin excrete high levels of dihydrodigoxin, an inactivemetabolite derived from reduction of an α,β-unsaturated lactone (112). Moreover, fecal samples from dihydrodigoxin-excreting individuals were found to completely metabolize digoxin. Subsequent isolation of digoxin-metabolizing microbes revealed that a single organism, Eubacterium lentum (renamed Eggerthella lenta), was responsible (113). However, E. lenta was also found in patients who did not excrete dihydrodigoxin, illustrating that the presence of this species alone was not predictive of activity. lactone . Early sE. lenta’s role in digoxinmetabolism has been appreciated for decades, challenges in growing the organism and a lack of genetic tools hampered efforts to understand this transformation. More recently, Turnbaugh and co-workers used RNA sequencing (RNA-seq) and comparative genomics to identify a digoxin-inducible gene cluster present only in digoxin-metabolizing E. lenta strains (37) (cgr) operon fromthis organism encodes two proteins that resemble bacterial reductases involved in anaerobic respiration. Bioinformatic analyses suggest that a membrane-associated cytochrome (Cgr1) transfers electrons through a series of hemes to a predicted flavin-dependent reductase (Cgr2) that converts digoxin to dihydrodigoxin. The presence of cgr genes correlates with digoxin reduction by E. lenta strains. Furthermore, experiments in GF mice monoassociated with E. lenta strains and incubations with human fecal samples indicate that these genes could be useful biomarkers for digoxin inactivation (37). Consequently, knowledge of the conditions influencing cgr gene expression successfully informed the design of a dietary intervention that reduces digoxin metabolism in vivo (37). Overall, this work delineated an approach that may be generalized to study additional inducible microbial metabolic activities. Although E. lenta’s role in digoxinmetabolism has been appreciated for decades, challenges in growing the organism and a lack of genetic tools hampered efforts to understand this transformation. More recently, Turnbaugh and co-workers used RNA sequencing (RNA-seq) and comparative genomics to identify a digoxin-inducible gene cluster present only in digoxin-metabolizing E. lenta strains (37) (cgr) operon fromthis organism encodes two proteins that resemble bacterial reductases involved in anaerobic respiration. Bioinformatic analyses suggest that a membrane-associated cytochrome (Cgr1) transfers electrons through a series of hemes to a predicted flavin-dependent reductase (Cgr2) that converts digoxin to dihydrodigoxin. The presence of cgr genes correlates with digoxin reduction by E. lenta strains. Furthermore, experiments in GF mice monoassociated with E. lenta strains and incubations with human fecal samples indicate that these genes could be useful biomarkers for digoxin inactivation (37). Consequently, knowledge of the conditions influencing cgr gene expression successfully informed the design of a dietary intervention that reduces digoxin metabolism in vivo (37). Overall, this work delineated an approach that may be generalized to study additional inducible microbial metabolic activities.Although ins (37) . The carins (37) . The carN-oxide (TMAO) by FMOs in the liver . A chemically guided, rational genome-mining effort ultimately identified the genes and enzymes that mediate this process (114). Choline fermentation begins with a C–N bond cleavage reaction that resembles the first step in ethanolamine utilization, a transformation catalyzed by a B12-dependent radical enzyme (ethanolamine ammonia-lyase). Hypothesizing that these pathways might share certain reactions, we searched the genome of the choline-metabolizing, animal-associated strain Desulfovibrio desulfuricans for homologs of known ethanolamine-degrading enzymes from the pathogen Salmonella enterica. This analysis revealed the choline utilization (cut) gene cluster and CutC, a GRE that converts choline into TMA and acetaldehyde .Gut microbes anaerobically convert choline, an essential nutrient found in meat, eggs, and milk, into TMA, which is further transformed into trimethylamine he liver . This co process  (114). C46, 115). These conserved amino acids are specific to this particular GRE and have enabled accurate identification of the cut pathway in numerous, phylogenetically diverse human gut bacteria (14). The discovery of CutC has aided interventions to modulate choline metabolism in vivo, including the design of gut communities with decreased TMA production (116) and small-molecule inhibitors targeting this pathway (117). Such inhibitors could be potential therapeutics to treat diseases linked to TMA and TMAO production.Biochemical and structural studies have revealed CutC active-site residues that interact with the trimethylammonium group of choline and mediate C–N bond cleavage (26). SN-38 then enters colonic epithelial cells, causing intestinal damage and severe diarrhea, side effects that limit the use of this otherwise effective drug.Irinotecan (CPT-11) is a prodrug of SN-38, a topoisomerase inhibitor used for treating cancer . SN-38 i26). These inhibitors were effective against multiple, distantly related gut bacteria but did not target the human b-glucuronidase. Structural studies revealed that these compounds interact with an active-site loop distinct to bacterial β-glucuronidases, which explains their selectivity. Administration of one of these inhibitors to mice prevented reactivation of SN-38 in the gut and concomitant toxicity. Further work examining the crystal structures and activities of additional gut bacterial β-glucuronidases identified a conserved Asn-Lysmotif that interacts with the carboxylic acid of the glucuronic acid sugar. This motif is absent from glycosidases that accept different substrates, which will help to identify these enzymes in sequencing data sets and elucidate the effects of inhibitors on different types of gut organisms (23). Because bacterial β-glucuronidases can deconjugate glucuronides derived from many dietary compounds and drugs, inhibitors of these enzymesmay be useful in other therapeutic contexts (27). By modulating specific metabolic activities, additional small-molecule inhibitors of microbial gut enzymes may help to uncover the roles of particular transformations in this complex habitat and could be excellent starting points for developingmicrobiotatargeted drugs.Inhibition of gut bacterial β-glucuronidases is an intriguing approach for preventing drug reactivation. As these enzymes are broadly distributed in commensal bacteria and are present in humans, inhibitors need to be selective for bacterial β-glucuronidases and nontoxic to both host cells and other gut microbes. Redinbo and coworkers have used an in vitro high-throughput screen to successfully identify potent and selective inhibitors of gut bacterial β-glucuronidases  (26). ThAlthough our appreciation of the human gut microbiota’s role in transforming xenobiotics has increased, our understanding of the biological implications of these reactions is limited. The future challenges lie in identifying the organisms, genes, and enzymes involved in known metabolic processes; uncovering important but currently unappreciated activities; and elucidating the effects of this chemistry on both host and microbiota.118), or culturing from human fecal samples can identify individual organisms with particular metabolic capabilities. Examining intact communities via approaches like stable isotope probing, fluorescence in situ hybridization, and imaging mass spectrometry can also be used to detect individual cells that metabolize particular compounds (119). Coupled with single-cell genomics, these methods may aid investigations of activities associated with organisms that are challenging to cultivate (120).As we have discussed above, most examples of gut microbial xenobiotic metabolism are associated with the whole gut community. Surveys of human gut isolates, such as Human Microbiome Project (HMP) reference strains . However, a lack of genetic tools for many gut microbes and the narrow range of heterologous hosts available for functional characterization still limit progress toward this objective. An appreciation of the chemical reactivity required for xenobiotic degradation is vital for informing genome and metagenome mining, as well as interpreting and rationalizing results from othermethods.Microbes in which enzymes and metabolic pathways are better studied, including pathogens and environmental strains involved in bioremediation, may also offer clues to support microbiome-focused discovery efforts.Both traditional approaches  and more recent strategies  may be used to link metabolic activities with genes (122).The examples of xenobiotic metabolism surveyed above likely represent a small fraction of the transformations taking place in the gut community. Metabolomics in human subjects will play a key role in illuminating currently unappreciated gut microbial metabolic processes, particularly for transformations that depend on additional microbial and host activities or interactions (10). Many members of large enzyme superfamilies also have unknown functions and are typically misannotated. Collaborative efforts such as the Enzyme Function Initiative have advanced bioinformatic and experimental approaches for functionally characterizing such enzymes (123), including protein sequence similarity and genome neighborhood network analyses, high-throughput ligand docking, and structural genomics. These methods highlight divergent sequences that likely possess distinct functions and provide context for linking uncharacterized enzymes withmetabolic pathways and substrates. Conversely, an understanding of the chemistry involved in known xenobioticmetabolism may pinpoint particular enzyme superfamilies as starting points to prospect for additional transformations.We should expect to uncover interesting gut microbial activities by investigating the vast number of uncharacterized or misannotated genes present in human gut microbiomes. Notably, 86% of the genes from HMP stool metagenomes cannot be assigned to known metabolic pathways, and half cannot be annotated .Comprehensively characterizing proteins that lack homology to known enzymes presents amore substantial challenge and will likely require novel approaches that incorporate automation and highthroughput assay formats. Meanwhile, it is critical to prioritize uncharacterized proteins for further study by considering both ecological context  and host health status, including exposure to xenobiotics .Diet is clearly a cornerstone of human health. Though many epidemiological studies linking dietary patterns with health outcomes yield conflicting results, few have taken gut microbial metabolic capabilities into account. A comprehensive molecular understanding of how gut microbes process dietary components is essential for the rational use of “functional foods” or prebiotics to treat conditions such as metabolic disease and malnutrition. This knowledge can also inform personalized nutrition, in which diets are individually customized for patients’ metabolic profiles and gut microbiotas  (125).126).Gut microbial enzymes that alter the toxicity of industrial chemicals and environmental pollutants could serve as biomarkers to inform risk assessments among populations exposed to these compounds (127).Similarly to dietary studies, attempts to correlate pollutant exposure with health outcomes often yield conflicting results that could, in part, arise from variations inmicrobial metabolic activities . Gut microbial enzymes that mediate harmful metabolic activities may also represent a new class of drug targets. Finally, knowledge of gutmicrobial metabolism could shape clinical trial design and clinical practice by allowing physicians to screen for detrimental or beneficial activities before prescribing drugs (Finally, knowledge of how gut microbes transformpharmaceuticals should be further integrated into drug development, clinical trial design, and clinical practice. Understanding which functional groups are prone to microbial metabolism allows medicinal chemists to either avoid these structural features or incorporate them into prodrugs to enable selective activation in the GI tract (ng drugs . UltimatA major challenge facing human gut microbiota research is the need to move beyond cataloging the organisms and genes in this community to elucidating the mechanisms underlying their influence on host health. By altering the chemical structures of ingested compounds, gut microbes can mediate the effects of diet, pollutants, and drugs on host physiology. Individual variation remains a major challenge, and although many such metabolic activities have been identified, few have been connected to organisms, genes, and enzymes.Moving forward, it is essential that we incorporate enzyme discovery and characterization efforts into investigations of gut microbial xenobiotic metabolism. Only by gaining a molecular understanding of these processes can we leverage the remarkable chemical abilities of this community to improve human health.


Telomeres are progressively eroded during repeated rounds of cell division due to the end replication problem but also undergo additional more substantial stochastic shortening events. In most cases, shortened telomeres induce a cell-cycle arrest or trigger apoptosis, although for those cells that bypass such signals during tumour progression, a critical length threshold is reached at which telomere dysfunction may ensue. Dysfunction of the telomere nucleoprotein complex can expose free chromosome ends to the DNA double-strand break (DSB) repair machinery, leading to telomere fusion with both telomeric and non-telomeric loci. The consequences of telomere fusions in promoting genome instability have long been appreciated through the breakage–fusion–bridge (BFB) cycle mechanism, although recent studies using high-throughput sequencing technologies have uncovered evidence of involvement in a wider spectrum of genomic rearrangements including chromothripsis. A critical step in cancer progression is the transition of a clone to immortality, through the stabilisation of the telomere repeat array. This can be achieved via the reactivation of telomerase, or the induction of the alternative lengthening of telomeres (ALT) pathway. Whilst telomere dysfunction may promote genome instability and tumour progression, by limiting the replicative potential of a cell and enforcing senescence, telomere shortening can act as a tumour suppressor mechanism. However, the burden of senescent cells has also been implicated as a driver of ageing and age-related pathology, and in the promotion of cancer through inflammatory signalling. Considering the critical role of telomere length in governing cancer biology, we review questions related to the prognostic value of studying the dynamics of telomere shortening and fusion, and discuss mechanisms and consequences of telomere-induced genome rearrangements. The higradation ,3,4. Thiradation ,6. This radation ,8,9,10.Due to the inability of the DNA replication machinery to fully replicate linear DNA (the end replication problem), telomeres progressively shorten with each cell division ,12. TeloIn most cells, very short telomeres lead to senescence and cell cycle arrest . In humaIn human somatic cells in which DNA damage checkpoints are compromised, continued cell division and consequent telomere loss past the point at which senescence normally occurs lead to a phase known as “crisis” that is characterised by telomere uncapping, subsequent fusion events, and widespread genome instability ,27. The This review is focused on telomere length dynamics in the context of cancer progression. We discuss how telomere dysfunction can lead to the evolution of an immortalised clonal population of cells with a radically altered karyotype and disrupted oncogene or tumour suppressor pathways. Additionally, the prognostic potential of telomere length or dysfunction is considered, and how this can be utilised to improve and personalise patient treatment course.Telomere length is a species-specific trait that shows considerable variability between individuals as well as between tissues and across different chromosome arms ,35,36. IThe length of telomeres is carefully regulated, with recent studies pointing to a telomere trimming mechanism involving telomeric zinc finger-associated protein (TZAP), which sets an upper limit on telomere length ,41. At sIn somatic stem cells and some immune cells, telomerase activity is detectable at low levels; however, this activity is clearly insufficient to maintain telomere length, as progressive telomere losses are observed during aging ,15,44. IIn vitro, subsets of telomeres have been observed with lengths far below the sample mean, consistent with a rapid deletion of the repeat array. These stochastic events were observed with a frequency of around 4% at the XpYp telomere resulting in telomeres with a length <2.32 standard deviations below the sample mean with potentially dysfunctional characteristics ,43. InteIn cell culture, telomere crisis can be induced by ablation of shelterin components such as TRF2, or by overexpression of a dominant-negative telomerase construct (DN-hTERT), followed by prolonged cell culture to induce replicative telomere erosion ,47. DN-hInitially, continual passage of cells that lack functional telomerase leads to progressive erosion of telomere length and a concomitant increase in the variance of the distribution consistent with random cell division and exponential growth . HoweverRecurrent point mutations in the promoter of TERT have been identified in a range of cancers, with an overall frequency of around 19%, although some cancers show a higher incidence ,56,57,58Telomere stabilisation by the ALT pathway relies on homologous recombination to elongate telomeres through a mechanism that is thought to resemble repair by the break-induced replication (BIR) pathway ,62,63,64The interplay of these two pathways is also beginning to be explored, with recent studies suggesting that telomerase may play an important role in ALT inhibition, with the implication that antitelomerase therapies may inadvertently promote ALT induction ,73. Telomeres that lack sufficient protection from the DSB repair machinery may undergo fusions with other telomeres in a head-to-head orientation or with other non-telomeric genomic loci following an additional genomic break. By studying the structure of telomere fusions at the nucleotide level, recent studies indicate that the classical and alternative non-homologous end joining pathways  play a central role in mediating these joins ,51,74,75The C-NHEJ pathway typically mediates blunt-end ligation, involving the high affinity binding of Ku proteins to either side of the break, which promotes interaction with the X-ray Cross Complementing: DNA ligase IV complex (XRCC4:LIG4) to catalyse the join ,76,77,78−/−, LIG4−/−). In the context of DN-hTERT overexpression to drive cells into crisis, wild-type and LIG4−/− cells displayed telomere fusions during crisis and readily escaped, though LIG4−/− cells displayed a reduction in the number of inter-chromosomal events between the 17p and XpYp telomere [−/− cells also displayed telomere fusions but were uniformly unable to escape crisis, with the entire culture perishing within two to three months [−/− cells showed a significantly higher proportion of inter-chromosomal events (17p to XpYp fusions) relative to wild-type and LIG4−/− cells.We have investigated the consequences of deficiencies in A- and C-NHEJ in mediating telomere fusions by the knockout of ligases LIG3 and LIG4  pathways may also play a role in telomere fusions. In cells lacking the shelterin components TRF2 and RAP1, the homologous recombination factors PARP1 and SLX4 promoted resection of telomeres resulting in widespread telomere-free fusions involving nearly 50% of chromosome ends . We hypoWhilst the end joining pathways that mediate telomere fusions are beginning to be elucidated, characterising the end-processing that occurs prior to repair has received less attention. From sequencing the break sites of end-to-end telomere fusions sister chromatids appeared to be differentially processed with one sister chromatid often undergoing a deletion event before joining ,51. The Telomere fusions have been shown to involve loci throughout the genome although the factors that govern where a fusion may occur remain poorly understood . There iComplex genome rearrangements can occur early during the progression to malignancy, preceding the invasive and metastatic stage of the disease, and are evident by the time telomerase expression is detected ,90,91. TDysfunctional telomeres can be fused with other loci in various configurations giving rise to sister chromatid, intra- or inter-chromosomal events. As replicative erosion occurs in parallel at all 92 telomeres of the cell, telomere dysfunction and telomere induced DNA damage signalling may occur rarely in isolation and instead may arise simultaneously at multiple ends. Additionally, the inception of telomere dysfunction may be most likely immediately following the telomere losses that occurs during DNA replication, when a dysfunctional telomere will be spatially prearranged with a sister chromatid with similar, potentially dysfunctional characteristics. Fusions between sister chromatids in end-to-end joining events are thus predicted to be more prevalent than inter-chromosomal end-to-end events, although limitations with current analysis techniques make this assumption difficult to prove ,87,93. IDicentric chromosome formation is one potential consequence of a telomere fusion event. They can be unstable structures that may be broken during cell division and initiate further rounds of fusion and breakage, leading to a cycle of events in what is known as the breakage-fusion-bridge (BFB) cycle ,95 Figu. DicentrThe genomic context of breakage during a BFB cycle is poorly defined, and may depend on several factors relating to the local chromatin environment through fragility effects, or may be related to sequence context or the presence of DNA lesions and modifications ,102. In Another possible consequence of a telomere fusion is the creation of a non-reciprocal translocation that results in the transfer of the distal portion of one chromosome arm to the dysfunctional telomere, or the partial copy of one chromosome by the break-induced replication pathway ,105. ThiHowever, several groups have recently raised the prospect that telomere-induced genome instability may have an immediate and even more spectacular consequence on the integrity of the genome, by instigating a genome catastrophe—a whole-genome restructuring event that occurs in a single step ,108,109 The promotion of large scale genome rearrangements and instability by telomere dysfunction has been well documented ,115,116.Chronic Lymphocytic Leukaemia (CLL) has provided an example of the use of telomere length measurements to define prognosis. CLL has a heterogeneous clinical course with overall survival ranging from a few months to many decades . A multiThe telomere length below which telomere fusion was detected was established in CLL B-cells. This “fusogenic” range was then used to stratify patients into favourable and unfavourable prognosis categories, even in early stage patients . PatientInterestingly, use of this fusogenic range prognostic tool also appears to successfully stratify patients into favourable and unfavourable disease progression categories in breast cancer . When a + plasma cell subsets with shorter telomere-length profiles, within the mixed population was sufficient to reduce mean telomere length and allow prognostic discrimination [Other cancers for which short telomere length can be used as a prognostic indicator include non-small-cell lung cancer, myelodysplastic syndromes (MDS) and multiple myeloma ,129,130.The above examples are consistent with the idea of telomere length loss and subsequent dysfunction as being a precursor to tumour progression. Other studies indicate that the converse may be true, where for some cancers, longer telomere length predicts poor clinical outcome ,136,137.Poorer clinical prognosis has been seen in patients with longer leukocyte telomere lengths for a number of cancers: in prostate cancer long telomere length was found to be an independent negative factor for both metastasis-free survival and PC-specific death when evaluated together with established risk factors  . SimilarU-shaped associations have also been observed between telomere length and cancer progression. In a recent case-control study in the Chinese population, both short and extremely long telomeres were found to be risk factors in oesophageal squamous cell carcinoma (ESCC) . A similThere is a clear need to accurately stratify cancer patients by clinical outlook as close to diagnosis as possible, thereby providing important information to patients, their clinicians and funding agencies, particularly with the advent of expensive targeted therapies. Telomere length shows great promise as a prognostic and predictive marker for certain cancers, though the sometimes contrasting results in telomere length association studies suggest a cautious outlook should be adopted. The influence of telomere length on driving oncogenesis may differ across cancer types, each with distinct phenotypes resulting in diverse effects on survival and modulating the prognostic potential of telomere length. Equally, study design parameters, experimental approach and analysis method may also influence the relationship between telomere length and cancer progression . SeveralCritically shortened telomeres, rendered dysfunctional by replicative erosion or stochastic deletion, play a central role in driving oncogenesis by generating genomic rearrangements that disrupt oncogene or tumour suppressor pathways, and by generating clonal diversity which fosters genome evolution through natural selection. Recent studies using high-throughput sequencing suggest that the complexity of telomere-induced genome instability may be have been greatly underappreciated, with extremely convoluted and intricate genomic configurations now being reported ,112,144.


Conduit geometry affects magma ascent dynamics and, consequently, the style and evolution of volcanic eruptions. However, despite geological evidences support the occurrence of conduit widening during most volcanic eruptions, the factors controlling conduit enlargement are still unclear, and the effects of syn-eruptive variations of conduit geometry have not been investigated in depth yet. Based on numerical modeling and the application of appropriate stability criteria, we found out a strong relationship between magma rheology and conduit stability, with significant effects on eruptive dynamics. Indeed, in order to be stable, conduits feeding dacitic/rhyolitic eruptions need larger diameters respect to their phonolitic/trachytic counterparts, resulting in the higher eruption rates commonly observed in dacitic/rhyolitic explosive events. Thus, in addition to magma source conditions and viscosity-dependent efficiency for outgassing, we suggest that typical eruption rates for different magma types are also controlled by conduit stability. Results are consistent with a compilation of volcanological data and selected case studies. As stability conditions are not uniform along the conduit, widening is expected to vary in depth, and three axisymmetric geometries with depth-dependent radii were investigated. They are able to produce major modifications in eruptive parameters, suggesting that eruptive dynamics is influenced by syn-eruptive changes in conduit geometry. Despite several numerical models have been employed for studying the physical conditions of ascending magmas6, they commonly assume mechanically stable geometries with constant radius in depth. Macedonio et al.7 identified four erosive mechanisms able to produce syn-eruptive changes in conduit geometry, indicating the zones where each one is probably dominant. Nonetheless, conditions for syn-eruptive mechanical stability of volcanic conduits have never been addressed in detail, nor has been studied the effect of magma rheology on them. Aravena et al.8 proposed that conduit dimensions control its mechanical stability, and a minimum radius (and hence a minimum mass flow rate) for reaching stable conditions can be computed. Under these assumptions, axisymmetric conduits with fixed radius represent a mechanically stable configuration only for a narrow range of conditions, and widening processes probably lead to geometries characterized by depth-dependent dimensions. Thus, since magma rheology controls the pressure profile along the conduit, which in turn controls the mechanical stability, important hints onto the relationships between magma composition, conduit stability and eruptive style are here derived by extending this analysis to different magma types commonly involved in large explosive events. In order to investigate this, we compare stability conditions for typical phonolitic, trachytic, dacitic and rhyolitic explosive eruptions. The most important eruptive variables and the pressure profile along the conduit (P(z)) were derived by using a 1D steady-state model9 (http://demichie.github.io/MAMMA) and, with the objective of determining the minimum pressure needed to avoid conduit collapse (Pcollapse(z)), we applied the Mogi – Coulomb collapse criterion10, as illustrated in Fig. Magma ascent dynamics during explosive eruptions is mainly controlled by the relation between magma supply and discharge ratecollapse(z) − P(z)), are preferably reached near and above the fragmentation level, a zone characterized by an abrupt pressure drop in the ascending magma 11. Hence, in addition to magma source conditions  and the viscosity-dependent efficiency of outgassing mechanisms (i.e. permeable degassing and bubbly flow), we suggest that typical eruption rates for different magma compositions are also controlled by conduit stability and its interplay with the changing properties of ascending magmas.Results indicate that magma rheology is a major controlling factor of the instability character of volcanic conduits. Indeed, in order to achieve stability conditions, dacitic/rhyolitic magmas need conduits several times wider than their phonolitic/trachytic counterparts; and significant differences are also observed between dacitic and rhyolitic melts. It is also interesting to note that, for dacitic and rhyolitic magmas, the stable conduits span over about just one order of magnitude, while they vary over about two orders of magnitude for phonolitic and trachytic magmas. In particular, for the range of input parameters here adopted and considering a 5000-m long conduit, critical radii range between 7 and 14 m for phonolitic magmas, whereas predicted critical radii for trachytic magmas vary between 6 and 13 m. Conversely, critical radii larger than 20 m and 50 m were estimated for dacitic and rhyolitic magmas, respectively. Hence, if we assume that volcanic conduits are continuously widened up to reach stability conditions, numerical simulations suggest that the ascending magma rheology controls the expected conduit dimensions and thus the resulting eruption rate. These results are consistent with the high mass discharge rates commonly observed in sustained dacitic/rhyolitic eruptions, whereas phonolitic/trachytic magmas are capable of producing sustained eruptions with narrower conduits and lower mass fluxes, as confirmed by a compilation of global data and the associated variance analysis , results suggest that the characteristic magma discharge rates needed for developing stationary explosive eruptions are highly controlled by magma rheology. Indeed, while quasi-steady, Plinian to sub-Plinian eruptions of phonolitic/trachytic magmas are often associated to intermediate values of mass discharge rate, steady dacitic/rhyolitic eruptions are generally characterized by very high magma discharge rates14, possibly stabilized by the development of wide conduits. Moreover, if collapsing conduits inhibit magma ascent, a minimum mass discharge rate for developing explosive eruptions is expected, significantly higher for dacitic/rhyolitic magmas than for their phonolitic/trachytic counterparts.Moreover, in order to produce a stable conduit, a minimum mass discharge rate can be derived for all the studied compositions  and gravitational collapse , in addition to the effect of other erosion mechanisms. This is indirectly confirmed by the ubiquitous presence of lithic fragments throughout all the deposits of large pyroclastic eruptions.Because of the strong contrast between the wall-rock volumes to erode for producing the different axisymmetric geometries with depth-dependent radii, important differences are expected on the composition and relative amount of lithic fragments in pyroclastic deposits. Indeed, the ratio between the mass of eroded lithic fragments and the eruption rate reveals that the expected lithic fraction with geometries NC2 and NC3 is one order of magnitude higher than that expected with geometry NC1, even though geometry NC1 produces more important effects on the eruptive dynamics. Thus, no direct relations between the observed fraction of lithic fragments in pyroclastic deposits and the magnitude of the eruptive dynamics variations are expected; and the effectiveness of wall rocks erosion processes in controlling the eruptive behavior is much higher for NC1-like geometries. Since axisymmetric geometries with depth-dependent radii do not necessarily tend to stability conditions6–6·106 kg/s)15 and fluctuations between magmatic and phreatomagmatic phases. Our results suggest that the low mass discharge rates produced unstable conditions in the conduit, generating episodic collapse events manifested in the pulsating nature of the eruption, possibly favoring the access of external water into the conduit. Indeed, since pore pressure favors conduit instabilities, collapse conditions were possibly concentrated around aquifer rocks12, producing magma-water interaction and the observed phreatomagmatic behavior during these pulsations. The range of mass discharge rate for this eruption is similar to that estimated for other sub-Plinian eruptions with magmas of similar rheology and characterized by an important unsteadiness17, suggesting a possible cause-effect relation with conduit stability conditions. In particular, the main part of the pyroclastic deposits related to the 512 AD phonolitic to tephri-phonolitic eruption of Vesuvius is characterized by a striking grain size alternation of fine to coarse lapilli, interpreted as the product of a highly oscillatory sub-Plinian phase16, followed by a pulsating phreatomagmatic phase. A similar unsteady behavior was interpreted for the initial sub-Plinian phase of the 472 AD eruption of Somma-Vesuvius (phonolitic to tephri-phonolitic magmas)17, with the alternating deposition of pyroclastic fall beds and pyroclastic density currents, also in this case followed by phases characterized by phreatomagmatic fragmentation.Data from selected eruptions can be used for a semi-quantitative validation of the modeling results. The trachytic 1630 eruption of Furnas volcano  represents an appropriate case study for illustrating the relation between mass discharge rate and the unsteady character of volcanic eruptions. It was a mainly pulsating event characterized by relatively low mass discharge rates  lithic-rich layers confined to pumice beds characterized by changes in the pumice size and/or evidences of interaction with external water, and interpreted as partial blockages of the conduit; and (2) lithic-rich layers in beds with uniform pumice size, indicating limited wall collapses, insufficient for interfering with the quasi-steady character of the Plinian eruption. The extent of conduit blockage related to wall collapse events is controlled by the collapsing volume and conduit dimensions. We suggest that, since narrow conduits produce favorable conditions to wall collapse (and consequent conduit blockage), the initial erosion rate (i.e. during inception and waxing phases) plays a crucial role in the viability of sustained Plinian eruptions. Indeed, seven units  out of twelve of Mangaone Subgroup are characterized by basal, lithic-rich deposits and peak mass discharge rates variable from 8.6·107 up to 3.9·108 kg/s. This clearly suggests that, in order to maintain a sustained flux of rhyolitic magma, an early conduit enlargement process is needed, and a consequence of this enlargement is the establishment of the high magma discharge rates typical of sustained Plinian eruptions18. On the other hand, the only two eruptions characterized by clear phases of collapsing column and ignimbrite deposition (units I and L), present a generally high lithic content throughout the entire sequence, suggesting that the continuous increase in conduit diameter was possibly one of the reasons for determining collapse conditions of the eruptive column. In addition, most of the units present lithic-rich beds during the waning phases and occasionally exhibit evidences of magma-water interaction18, interpreted as the result of incipient wall collapses produced by inlet pressure drops, before the total blockage of the conduit (i.e. shutdown of the eruption). Taupo pumice represents an additional case study for addressing sustained silicic eruptions19. In this case, the reported eruption rate is higher than 109 kg/s and, in agreement with our results, it produced a sustained explosive event. Well-constrained values of total lithic content (1.8·1012 kg)19 provide some lights for estimating feeding conduit dimensions. Considering an 8000-m long axisymmetric conduit with fixed dimensions in depth, radii larger than 170 m are expected, producing a highly stable conduit according to our results. Numerical simulations suggest that such particularly large conduit diameter could be related to an efficient conduit-opening process, probably favored by magma-water interaction12, with significant consequences on the resulting mass discharge rate. Additional suitable information is provided by the dacitic-rhyodacitic 1932 Quizapu eruption (Chilean Andes), which produced an “exceptionally uniform fall deposit”20. Still, as observed in most of the rhyolitic Mangaone eruptions, also in this case the lower part of the deposit is richer in lithic fragments than the upper part, and a general  upward coarsening of the fall deposit is described. Despite the percentage of lithic fragments is low, because of the high erupted volume, it resulted in a large volume of lithic fragments20, implying conduit radii larger than 100 m. These conduit dimensions and the reported mass discharge rate (9·107–2.5·108 kg/s)20 are in agreement with the development of a very stable feeding conduit and a highly steady explosive eruption, manifested in the described uniformity of pyroclastic deposits20.The detailed, systematic study of the rhyolitic eruptions of the Mangaone subgroup 23. During the latest explosive event in the Inyo volcanic chain, ~0.13 km3 of rhyolitic tephra was erupted in four successive eruptions. The first two eruptions produced pulsating, sub-Plinian columns separated by a period of column collapse22, exhibiting mass discharge rates lower than 107 kg/s, which is in agreement with the highly unstable character expected for these MDRs in rhyolitic/rhyodacitic feeding conduits. The latter two eruptions were characterized by higher MDRs , and the development of more sustained weak plumes, still with punctual periods of phreatomagmatic activity22. The transition between the second (South Deadman 2) and third eruption (Obsidian flow) was marked by a phreatomagmatic vent-clearing blast22, which would have suddenly increased conduit dimensions, favoring stability conditions and the development of more steady explosive events, in comparison with the two first eruptions. For Inyo Craters, geological evidences strongly support that magma ascent is particularly favored by structural settings25, thus it seems difficult to use lithic content to estimate conduit dimensions. Still, even in the latter two eruptions, total volumes of lithic fragments  provide a subset of silicic explosive eruptions characterized by relatively low eruption ratesDacitic/rhyolitic magmas need conduits several times wider than phonolitic/trachytic magmas for developing mechanically stable conduits, which could explain the high mass discharge rates commonly observed in sustained dacitic/rhyolitic eruptions, whereas phonolitic/trachytic magmas would be capable of producing sustained explosive eruptions with relatively low mass discharge rates.Conduit stability can influence the steadiness of volcanic eruptions, controlling the occurrence of pulsating variations in the eruptive parameters. Narrow, unstable conduits would be commonly related with oscillating volcanic eruptions, whereas wide, stable feeding conduits could produce steady explosive events and homogeneous pyroclastic deposits.Conduit wall collapses seem to be particularly dominant during inception/waxing phases of explosive events, determining whether a well-developed steady-state phase is reached and thus controlling the early dynamics of volcanic events and the viability of sustained eruptions. Conduit collapse is also likely to occur during the waning phases of explosive eruptions, because of the expected inlet pressure drop, generating conduit blockages and controlling the eruption shutdown.The nature of the erosive process along the conduit during volcanic eruptions is a first order factor for controlling the resulting eruptive dynamics, modifying parameters such as mass discharge rate, exit pressure and fragmentation depth. In particular, the eruptive dynamics would be conditioned by the ability of the conduit to inhibit widening processes above the fragmentation level.Modeling of magma ascent through axisymmetric volcanic conduits with fixed and depth-dependent radii suggests that magma composition and rheology play a leading role in defining the conditions of conduit stability, and consequently in controlling conduit dimensions, evolution of mass discharge rate and the amount of lithic fragments found in pyroclastic deposits. In particular:In order to study the pressure field along volcanic conduits (P(z)), we employed a 1D multiphase steady-state model which considers the main processes experienced by magmas during their ascent, such as crystallization, rheological changes, magma fragmentation, interaction with conduit walls, outgassing and degassing. We used appropriate sets of constitutive equations for describing the physical parameters of the system (Supplementary Table collapse(z)), as described in Aravena et al.8. For that, we applied the Mogi-Coulomb criterion presented by Al-Ajmi and Zimmerman10 (Supplementary Table collapse(z) and P(z), using the following expression, called instability index8:For evaluating the mechanical stability of the conduit, we calculated the minimum pressure needed to avoid conduit collapse , using a second order fit. Accordingly, the critical radius represents the minimum radius needed to avoid conduit collapse, for a given set of magma source conditions.Since a monotonic relationship is observed between conduit radius and instability index11 kg and 2·1012 kg for trachytic and rhyolitic magmas, respectively. This assumption is supported in the absence of exsolved water in magma reservoir for the solubility models here adopted (conduit length of 5000 m), and thus nearly incompressible melts are expected to fill the magma reservoir. For a given magma composition, two different hypothetical events were modelled, which are characterized by two different prescribed mathematical relations between inlet pressure and water content, and reflect discrepancies in compositional zonations of magma chamber , as a function of the mass of erupted magma; considering a total erupted mass of 10ber Fig. . Still, r (geometry NC1), (3) conduits with two coaxial cylindrical portions connected by a linearly variable transitional zone of length zr (geometry NC2) and (4) conduits with an axisymmetric lower portion with fixed radius in depth, connected with the surface by a linearly enlarging zone (geometry NC3). In simulations of axisymmetric conduits with depth-dependent radius , we set the variable-radius zone (zc) around the fragmentation level computed in the fixed-radius equivalent case (i.e. Rfixed(C) = Rmin(NC), with the same values of inlet pressure and water content); varying Rmax/Rmin and zr between specific limits  axisymmetric conduits with fixed radius in depth (geometry C), (2) axisymmetric fixed-radius conduits with a local enlargement along a vertical distance z26. As an intensity measure, we employed the eruptive column height11, splitting data into four subsets: (1) basaltic and andesitic basaltic magmas, (2) andesitic magmas, (3) dacitic and rhyolitic magmas and (4) phonolitic and trachytic magmas. When two or more results are reported for different phases of a unique eruption, we selected the highest value; and we discarded all data with other compositions or information difficult to split . Descriptive statistics and variance analysis were employed for results interpretation.On the other hand, in order to compare the typical intensity of explosive volcanic eruptions related to different magma compositions, we used information derived from a global database (LaMEVE)Supplementary material