An integrated assessment of the 1,4-dioxane cancer mode of action and threshold response in rodents.

1,4-Dioxane is an environmental contaminant that has been shown to cause cancer in rodents after chronic high dose exposures. We reviewed and integrated information from recently published studies to update our understanding of the cancer mode of action of 1,4-dioxane. Tumor development in rodents from exposure to high doses of 1,4-dioxane is preceded by pre-neoplastic events including increased hepatic genomic signaling activity related to mitogenesis, elevation of Cyp2E1 activity and oxidative stress leading to genotoxicity and cytotoxicity. These events are followed by regenerative repair and proliferation and eventual development of tumors. Importantly, these events occur at doses that exceed the metabolic clearance of absorbed 1,4-dioxane in rats and mice resulting in elevated systemic levels of parent 1,4-dioxane. Consistent with previous reviews, we found no evidence of direct mutagenicity from exposure to 1,4-dioxane. We also found no evidence of CAR/PXR, AhR or PPARα activation resulting from exposure to 1,4-dioxane. This integrated assessment supports a cancer mode of action that is dependent on exceeding the metabolic clearance of absorbed 1,4-dioxane, direct mitogenesis, elevation of Cyp2E1 activity and oxidative stress leading to genotoxicity and cytotoxicity followed by sustained proliferation driven by regenerative repair and progression of heritable lesions to tumor development.

Mode of action assessment for propylene dichloride as a human carcinogen.

As part of a systematic review of the non-cancer and cancer hazards of propylene dichloride (PDC), with a focus on potential carcinogenicity in workers following inhalation exposures, we determined that a mode of action (MOA)-centric framing of cancer effects was warranted. In our MOA analysis, we systematically reviewed the available mechanistic evidence for PDC-induced carcinogenesis, and we mapped biologically plausible MOA pathways and key events (KEs), as guided by the International Programme on Chemical Safety (IPCS)-MOA framework. For the identified pathways and KEs, biological concordance, essentiality of KEs, concordance of empirical observations among KEs, consistency, and analogy were evaluated. The results of this analysis indicate that multiple biologically plausible pathways may contribute to the cancer MOA for PDC, but that the relevant pathways vary by exposure route and level, tissue type, and species; further, more than one pathway may occur concurrently at high exposure levels. While several important data gaps exist, evidence from in vitro mechanistic studies, in vivo experimental animal studies, and ex vivo human tumor tissue analyses indicates that the predominant MOA pathway likely involves saturation of cytochrome p450 2E1 (CYP2E1)-glutathione (GSH) detoxification (molecular initiating event; MIE), accumulation of CYP2E1-oxidative metabolites, cytotoxicity, chronic tissue damage and inflammation, and ultimately tumor formation. Tumors may occur through several subsets of inflammatory KEs, including inflammation-induced aberrant expression of activation-induced cytidine deaminase (AID), which causes DNA strand breaks and mutations and can lead to tumors with a characteristic mutational signature found in occupational cholangiocarcinoma. Dose concordance analysis showed that low-dose mutagenicity (from any pathway) is not a driving MOA, and that prevention of target tissue damage and inflammation (associated with saturation of CYP2E1-GSH detoxification) is expected to also prevent the cascade of processes responsible for tumor formation.

A 90-day drinking water study in mice to characterize early events in the cancer mode of action of 1,4-dioxane.

Studies demonstrate that with sufficient dose and duration, 1,4-dioxane (1,4-DX) induces liver tumors in laboratory rodent models. The available evidence aligns with a threshold-dependent, tumor promotion mode of action (MOA). The MOA and key events (KE) in rats are well developed but less so in the mouse. Therefore, we conducted a 90-day drinking water study in female mice to evaluate early KE at 7, 28, and 90 days. Female B6D2F1/Crl mice consumed drinking water containing 0, 40, 200, 600, 2000 or 6000 ppm 1,4-DX. 1,4-DX was detected in blood at 90-days of exposure to 6000 ppm, but not in the other exposure groups, indicating a metabolic clearance threshold between 2000 and 6000. Early events identified in this study include glycogen-like vacuolization, centrilobular hypertrophy, centrilobular GST-P staining, apoptosis, and pan-lobular increase in cell proliferation observed after 90-days of exposure to 6000 ppm 1,4-DX. There was minimal evidence of hepatotoxicity over the duration of this study. These findings demonstrate a previously unreported direct mitogenic response following exposures exceeding the metabolic clearance threshold of 1,4-DX. Collectively, the information generated in this study supports a threshold MOA for the development of liver tumors in mice after exposure to 1,4-DX.

Considerations for the Use of Mutation as a Regulatory Endpoint in Risk Assessment.

Assessment of a chemical's potential to cause permanent changes in the genetic code has been a common practice in the industry and regulatory settings for decades. Furthermore, the genetic toxicity battery of tests has typically been employed during the earliest stages of the research and development programs of new product development. A positive outcome from such battery has a major impact on the chemical's utility, industrial hygiene, product stewardship practices, and product life cycle analysis, among many other decisions that need to be taken by the industry, even before the registration of a chemical is undertaken. Under the prevailing regulatory paradigm, the dichotomous (yes/no) evaluation of the chemical's genotoxic potential leads to a conservative, linear no-threshold (LNT) risk assessment, unless compelling and undeniable data to the contrary can be provided to satisfy regulators, typically in a number of different global jurisdictions. With the current advent of predictive methods, new testing paradigms, mode-of-action/adverse outcome pathways, and quantitative risk assessment approaches, various stakeholders are starting to employ these state-of-the-science methodologies to further the conversation on decision making and advance the regulatory paradigm beyond the dominant LNT status quo. This commentary describes these novel methodologies, relevant biological responses, and how these can affect internal and regulatory risk assessment approaches. Environ. Mol. Mutagen. 61:84-93, 2020. © 2019 Wiley Periodicals, Inc.

A highly sensitive and selective high pressure liquid chromatography with tandem mass spectrometry (HPLC/MS-MS) method for the direct peptide reactivity assay (DPRA).

While the HPLC/UV (high performance liquid chromatography coupled with ultra-violet spectrometry)-based DPRA (Direct Peptide Reactivity Assay) identifies dermal sensitizers with approximately 80% accuracy, the low selectivity and sensitivity of the HPLC/UV-based DPRA poses challenges to accurately identify the sensitization potential of certain chemicals. In this study, a high performance liquid chromatography coupled with tandem mass spectrometry (HPLC/MS-MS)-based DPRA was developed and validated according to the test guideline (OECD TG 442C). The final results were compared with the results from the traditional HPLC/UV-based guideline DPRA. This HPLC/MS-MS-based DPRA demonstrated similar performance compared to HPLC/UV-based DPRA using known dermal sensitizers and non-sensitizers according to the test guideline (OECD TG 442C). Following the validation, a challenge set of chemicals with either overlapping retention time with peptides, or higher hydrophobicity or chemicals potentially forming non-covalent interactions with peptides were assessed for dermal sensitization potential using both methods and the results were compared to existing in vivo data. The HPLC/MS-MS-based DPRA correctly predicted these chemicals as sensitizers or non-sensitizers; however, the HPLC/UV-based DPRA resulted in false-positive predictions for hydrophobic substances, chemicals with UV peaks overlapping with those of the peptide(s), and compounds that non-covalently interact with the peptides. These findings demonstrate the broader applicability and better sensitivity and selectivity of the LC/MS-MS-based DPRA over the traditional HPLC/UV-based guideline DPRA.

A toxicological review of the ethylene glycol series: Commonalities and differences in toxicity and modes of action.

This review summarizes the hazards, exposure and risk that are associated with ethylene glycols (EGs) in their intended applications. Ethylene glycol (EG; CAS RN 107-21-1) and its related oligomers include mono-, di-, tri-, tetra-, and penta-EG. All of the EGs are quickly and extensively absorbed following ingestion and inhalation, but not by the dermal route. Metabolism involves oxidation to the mono- and dicarboxylic acids. Elimination is primarily through the urine as the parent compound or the monoacid, and, in the case of EG, also as exhaled carbon dioxide. All EGs exert acute toxicity in a similar manner, characterized by CNS depression and metabolic acidosis in humans and rodents; the larger molecules being proportionally less acutely toxic on a strict mg/kg basis. Species differences exist in the metabolism and distribution of toxic metabolites, particularly with the formation of glycolic acids and oxalates (OX) from EG and diethylene glycol (DEG); OX are not formed to a significant degree in higher ethylene glycols. Among rodents, rats are more sensitive than mice, and males more sensitive than females to the acute and repeated-dose toxicity of EG. The metabolic formation of glycolic acid (GA), diglycolic acid (DGA), and OX are associated with nephrotoxicity in humans and rodents following single and repeated exposures. However, physiological and metabolic differences in the rate of formation of GA, DGA and OX and their distribution result in EG and DEG causing embryotoxicity in rats, but not rabbits. This rodent-specific sensitivity indicates that EG and its higher oligomers are not anticipated to be embryotoxic in humans at environmentally relevant doses. None of the compounds present developmental toxicity concerns at doses that do not also cause significant maternal toxicity, nor do any of the EGs cause adverse effects on fertility. The EGs are neither genotoxic nor carcinogenic. A read-across matrix is presented, which considers the common and distinct toxicological properties of each compound. It is concluded that EGs pose no risk to human health as a result of their intended use patterns.

Human health assessment for long-term oral ingestion of diethylene glycol.

Diethylene glycol (DEG) is an organic chemical that is used mostly as a chemical intermediate and has minor uses as a solvent or antifreeze in consumer products; these minor uses could result in potential human exposure. Potential short and long-term human exposures also occur from misuses. The considerable reporting of DEG misuses as a substitute for other solvents in drug manufacturing and summaries of important events in the history of DEG poisonings are reviewed. Given the potential for human exposure, the disposition and toxicity of DEG were examined, and a health assessment was performed. Toxicokinetics and metabolism studies are evaluated, along with a discussion on the renal toxicity mode of action in the rat. Additionally, in-depth assessments of the key animal research studies on the toxic effects of DEG from oral ingestion for various exposure time periods are presented with determination of NOAELs and LOAELs from the long-term exposure animal studies. These are applied in the derivation of a reference dose for a non-cancer endpoint from chronic exposure, resulting in a value of 0.3 mg DEG/kg bw.

Contributions of DNA repair and damage response pathways to the non-linear genotoxic responses of alkylating agents.

From a risk assessment perspective, DNA-reactive agents are conventionally assumed to have genotoxic risks at all exposure levels, thus applying a linear extrapolation for low-dose responses. New approaches discussed here, including more diverse and sensitive methods for assessing DNA damage and DNA repair, strongly support the existence of measurable regions where genotoxic responses with increasing doses are insignificant relative to control. Model monofunctional alkylating agents have in vitro and in vivo datasets amenable to determination of points of departure (PoDs) for genotoxic effects. A session at the 2013 Society of Toxicology meeting provided an opportunity to survey the progress in understanding the biological basis of empirically-observed PoDs for DNA alkylating agents. Together with the literature published since, this review discusses cellular pathways activated by endogenous and exogenous alkylation DNA damage. Cells have evolved conserved processes that monitor and counteract a spontaneous steady-state level of DNA damage. The ubiquitous network of DNA repair pathways serves as the first line of defense for clearing of the DNA damage and preventing mutation. Other biological pathways discussed here that are activated by genotoxic stress include post-translational activation of cell cycle networks and transcriptional networks for apoptosis/cell death. The interactions of various DNA repair and DNA damage response pathways provide biological bases for the observed PoD behaviors seen with genotoxic compounds. Thus, after formation of DNA adducts, the activation of cellular pathways can lead to the avoidance of a mutagenic outcome. The understanding of the cellular mechanisms acting within the low-dose region will serve to better characterize risks from exposures to DNA-reactive agents at environmentally-relevant concentrations.

Repair of endogenous DNA base lesions modulate lifespan in mice.

The accumulation of DNA damage is thought to contribute to the physiological decay associated with the aging process. Here, we report the results of a large-scale study examining longevity in various mouse models defective in the repair of DNA alkylation damage, or defective in the DNA damage response. We find that the repair of spontaneous DNA damage by alkyladenine DNA glycosylase (Aag/Mpg)-initiated base excision repair and O(6)-methylguanine DNA methyltransferase (Mgmt)-mediated direct reversal contributes to maximum life span in the laboratory mouse. We also uncovered important genetic interactions between Aag, which excises a wide variety of damaged DNA bases, and the DNA damage sensor and signaling protein, Atm. We show that Atm plays a role in mediating survival in the face of both spontaneous and induced DNA damage, and that Aag deficiency not only promotes overall survival, but also alters the tumor spectrum in Atm(-/-) mice. Further, the reversal of spontaneous alkylation damage by Mgmt interacts with the DNA mismatch repair pathway to modulate survival and tumor spectrum. Since these aging studies were performed without treatment with DNA damaging agents, our results indicate that the DNA damage that is generated endogenously accumulates with age, and that DNA alkylation repair proteins play a role in influencing longevity.

The distribution, fate, and effects of propylene glycol substances in the environment.

The propylene glycol substances comprise a homologous family of synthetic organic molecules that have widespread use and very high production volumes across the globe. The information presented and summarized here is intended to provide an overview of the most current and reliable information available for assessing the potential environmental exposures and impacts of these substances across the manufacture, use, and disposal phases of their product life cycles.The PG substances are characterized as being miscible in water, having very low octanol-water partition coefficients (log Pow) and exhibiting low potential to volatilize from water or soil in both pure and dissolved forms. The combination of these properties dictates that, almost regardless of the mode of their initial emission, they will ultimately associate with surface water, soil, and the related groundwater compartments in the environment. These substances have low affinity for soil and sediment particles, and thus will remain mobile and bio-available within these media.In the atmosphere, the PG substances are demonstrated to have short lifetimes(I. 7-11 h), due to rapid reaction with photochemically-generated hydroxyl radicals.This reactivity, combined with efficient wet deposition of their vapor and aerosol forms, lends to their very low potential for long-range transport via the atmosphere.In the aquatic and terrestrial compartments of the environment, the PG substances are rapidly and ultimately biodegraded under both aerobic and anaerobic conditions by a wide variety of microorganisms, regardless of prior adaptation to the substances.Except for the TePG substance, the propylene glycol substances meet the OECD definition of "readily biodegradable", and according to this definition are not expected to persist in either aquatic or terrestrial environments. The TePG exhibits inherent biodegradability, is not regarded to be persistent, and is expected to ultimately biodegrade in the environment, albeit at a somewhat slower rate. The apparent ease with which microorganisms and higher organisms can metabolize the PG substances, along with their low log Pow and very high water solubility values, portends them to have very low potential for bioaccumulation and/or biomagnification in aquatic and terrestrial organisms. These same properties, along with their neutral structures and lack of biological reactivity, are the reasons for which the PG substances exhibit a base-line, non-polar narcosis mode of toxicity.The PG substances have been shown here to be practically non-toxic to essentially every aquatic and terrestrial animal and plant species tested. Collectively, the available wealth of information relating to persistence, bioaccumulation, and eco-toxicity of these substances allows a definitive conclusion of their categorization as not being PBT (i.e., persistently bioaccumulative/toxic). The PBT screening and categorization of substances on the Canadian Domestic Substances List (DSL) by Environment Canada has formally concluded that each member of this substance family is "not P", "not B", and "not T' according to their associated PBT criteria.Similarly, the preceding evaluations of these high production volume substances within the OECD SIDS program concluded that MPG, DPG, and TPG are low priorities for further examination of potential impacts to humans and the environment.More extensive evaluations of potential risks to human health and the environment were recently completed by industry, as required for their registration under the European Union REACh legislation; each evaluation demonstrated that current uses, associated exposures, and controls thereof, will not result in exposures that exceed predicted no effect concentrations in the environment.

The challenge of using read-across within the EU REACH regulatory framework; how much uncertainty is too much? Dipropylene glycol methyl ether acetate, an exemplary case study.

The use of read-across of data within a group of structurally similar substances potentially allows one to characterise the hazards of a substance without resorting to additional animal studies. However the use of read-across is not without challenges, particularly when used to address the needs of a regulatory programme such as the EU REACH regulation. This paper presents a case study where a previously accepted read-across approach was used to address several data gaps in a REACH registration dossier but was subsequently rejected in part by the European Chemicals Agency (ECHA), resulting in the requirement to perform a developmental toxicity study in rodents. Using this case study, this paper illustrates some of the practical challenges faced when making use of read-across, particularly with respect to addressing the uncertainty associated with the use of read-across; showcasing the scientific justification and highlighting some of the potential implications/opportunities for future cases.

Epigenetic screening in product safety assessment: are we there yet?

There has been a growing concern that epigenetic events, that is, heritable changes in gene expression superimposed on DNA nucleotide sequences, may be involved in chemically and/or nutritionally mediated adverse health outcomes, such as reproductive toxicity and cancer. This concern has been driven by an increasing number of studies reporting toxicant-induced alterations to the epigenome in the form of changes in DNA methylation, histone/chromatin remodeling, and altered expression of non-coding RNAs. These three major mechanisms of epigenetic modifications may have coordinated, independent, or potentially antagonistic influences on gene expression. Complicating this understanding is the incomplete understanding of the normal state and dynamic variation of the epigenome, which differs widely between cells, tissues, developmental state, age, strain, and species. This review serves as a framework to outline characteristics composing an ideal epigenetic screen(s) for hazard identification in product safety assessment. In order to implement such a screen, first there needs to be a better understanding of adaptive versus adverse changes in the epigenome, which includes identification of robust and reproducible causal links between epigenetic changes and adverse apical end points, and second development of improved reporter assay tools to monitor such changes. An ideal screen would be in vitro-based, medium- to high-throughput, and assess all three branches of epigenome control (i.e. methylation, histone modifications, non-coding RNAs), although also being quantitative, objective, portable (i.e. lab to lab), and relevant to humans.

Epigenetics and chemical safety assessment.

Epigenetics, as it pertains to biology and toxicology, can be defined as heritable changes in gene expression that do not involve mutations and are propagated without continued stimulus. Although potentially reversible, these heritable changes may be classified as mitotic, meiotic, or transgenerational, implicating the wide-ranging impact of epigenetic control in cellular function. A number of biological responses have been classified as being caused by an "epigenetic alteration," sometimes based on sound scientific evidence and often in lieu of an identified genetic mutation. Complicating the understanding and interpretation of perceived epigenetic alterations is an incomplete understanding of the normal state and dynamic variation of the epigenome, which can differ widely between cell and tissue types and stage of development or age. This emerging field is likely to have a profound impact on the study and practice of toxicology in coming years. This document reviews the current state of the science in epigenetic modifications, techniques used to measure these changes, and evaluates the current toxicology testing battery with respect to strengths and potential weaknesses in the identification of epigenetics changes. In addition, case studies implicating transgenerational effects induced by diethylstilbestrol, vinclozolin, and bisphenol A were reviewed to illustrate the application of epigenetics in safety assessment and the strengths and limitations of the study designs. An assessment of toxicology tests currently used in safety evaluation revealed that these tests are expected to identify any potential adverse outcomes resulting from epigenetic changes. Furthermore, in order to increase our understanding of the science of epigenetics in toxicology, this review has revealed that a solid understanding of the biology and variation in the epigenome is essential to contextualize concerns about possible adverse health effects related to epigenetic changes. Finally, the fundamental principles guiding toxicology studies, including relevant doses, dose-rates, routes of exposure, and experimental models, need to be taken into consideration in the design and interpretation of studies within this emerging area of science.

Frameshift mutagenesis and microsatellite instability induced by human alkyladenine DNA glycosylase.

Human alkyladenine DNA glycosylase (hAAG) excises alkylated purines, hypoxanthine, and etheno bases from DNA to form abasic (AP) sites. Surprisingly, elevated expression of hAAG increases spontaneous frameshift mutagenesis. By random mutagenesis of eight active site residues, we isolated hAAG-Y127I/H136L double mutant that induces even higher rates of frameshift mutation than does the wild-type hAAG; the Y127I mutation accounts for the majority of the hAAG-Y127I/H136L-induced mutator phenotype. The hAAG-Y127I/H136L and hAAG-Y127I mutants increased the rate of spontaneous frameshifts by up to 120-fold in S. cerevisiae and also induced high rates of microsatellite instability (MSI) in human cells. hAAG and its mutants bind DNA containing one and two base-pair loops with significant affinity, thus shielding them from mismatch repair; the strength of such binding correlates with their ability to induce the mutator phenotype. This study provides important insights into the mechanism of hAAG-induced genomic instability.

O6-methylguanine-induced cell death involves exonuclease 1 as well as DNA mismatch recognition in vivo.

Alkylation-induced O(6)-methylguanine (O(6)MeG) DNA lesions can be mutagenic or cytotoxic if unrepaired by the O(6)MeG-DNA methyltransferase (Mgmt) protein. O(6)MeG pairs with T during DNA replication, and if the O(6)MeG:T mismatch persists, a G:C to A:T transition mutation is fixed at the next replication cycle. O(6)MeG:T mismatch detection by MutSalpha and MutLalpha leads to apoptotic cell death, but the mechanism by which this occurs has been elusive. To explore how mismatch repair mediates O(6)MeG-dependent apoptosis, we used an Mgmt-null mouse model combined with either the Msh6-null mutant (defective in mismatch recognition) or the Exo1-null mutant (impaired in the excision step of mismatch repair). Mouse embryonic fibroblasts and bone marrow cells derived from Mgmt-null mice were much more alkylation-sensitive than wild type, as expected. However, ablation of either Msh6 or Exo1 function rendered these Mgmt-null cells just as resistant to alkylation-induced cytotoxicity as wild-type cells. Rapidly proliferating tissues in Mgmt-null mice (bone marrow, thymus, and spleen) are extremely sensitive to apoptosis induced by O(6)MeG-producing agents. Here, we show that ablation of either Msh6 or Exo1 function in the Mgmt-null mouse renders these rapidly proliferating tissues alkylation-resistant. However, whereas the Msh6 defect confers total alkylation resistance, the Exo1 defect leads to a variable tissue-specific alkylation resistance phenotype. Our results indicate that Exo1 plays an important role in the induction of apoptosis by unrepaired O(6)MeGs.

Transcriptional networks in S. cerevisiae linked to an accumulation of base excision repair intermediates.

Upon exposure to DNA damaging agents, Saccharomyces cerevisiae respond by activating a massive transcriptional program that reflects the fact that "DNA damaging" agents also damage other cellular macromolecules. To identify the transcriptional response that is specific to DNA damage, we have modulated the first two enzymes in the base excision repair (BER) pathway generating yeast strains with varied levels of the repair intermediates, abasic sites or strand breaks. We show that the number of abasic sites is significantly increased when the 3-methyladenine DNA glycosylase (Mag): AP endonuclease (Apn1) ratio is increased and that spontaneous frame shift mutation is considerably elevated when either Mag, or Mag plus Apn1, expression is elevated. Expression profiling identified 633 ORFs with differential expression associated with BER modulation. Analysis of transcriptional networks associated with the accumulation of DNA repair intermediates identifies an enrichment for numerous biological processes. Moreover, most of the BER-activated transcriptional response was independent of the classical yeast environmental stress response (ESR). This study highlights that DNA damage in the form of abasic sites or strand breaks resulting from BER modulation is a trigger for substantial genome-wide change and that this response is largely ESR-independent. Taken together, these results suggest that a branch point exists in the current model for DNA damage-signaled transcription in S. cerevisiae.

Transcription promotes guanine to thymine mutations in the non-transcribed strand of an Escherichia coli gene.

Transcription of DNA opens the chromatin, causes topological changes in DNA and transiently exposes the two strands to different biochemical environments. Consequently, it has long been argued that transcription may promote damage to DNA and there are data in Escherichia coli and yeast supporting a correlation between high transcription and mutations. We examined the transcription-dependence of the reversion of a nonsense codon (TGA) in E. coli and found that there was a strong dependence of mutations on transcription in strains defective in the repair of 8-oxoguanine in DNA. Under conditions of high transcription there was a three to five-fold increase in mutations that changed TGA in the non-transcribed strand to a sense codon. Furthermore, in both mutY and mutM mutY backgrounds the mutations were overwhelmingly G:C to T:A. In contrast, when the TGA was in the transcribed strand in relation with the inducible promoter, high transcription decreased the rate of reversion. Similar results were obtained in a strain defective in the transcription-repair coupling factor, Mfd, suggesting that transcription dependent increase in base substitutions does not require transcription-dependent DNA repair. However, Mfd does modulate the magnitude of the mutagenic effect of transcription. These data are consistent with a model in which the non-transcribed strand is more susceptible to oxidative damage during transcription than the transcribed strand. These results suggest that the magnitudes of individual base substitutions and their relative numbers in other studies of mutational spectra may also be affected by transcription.

Human activation-induced cytidine deaminase causes transcription-dependent, strand-biased C to U deaminations.

Activation-induced cytidine deaminase (AID) is required for the maturation of antibodies in higher vertebrates, where it promotes somatic hypermutation (SHM), class switch recombination and gene conversion. While it is known that SHM requires high levels of transcription of the target genes, it is unclear whether this is because AID targets transcribed genes. We show here that the human AID promotes C to T mutations in Escherichia coli which are stimulated by transcription. The mutations are strand-biased and occur preferentially in the non-transcribed strand of the target gene. Human AID purified from E.coli is active without prior treatment with a ribonuclease and deaminates cytosines in plasmid DNA in vitro. Further, the action of this enzyme is greatly stimulated by the transcription of the target gene in a strand-dependent fashion. These results confirm the prediction that AID may act directly on DNA and show that it can act on transcribing DNA in the absence of specialized DNA structures such as R-loops. It suggests that AID may be recruited to variable genes through transcription without the assistance of other proteins and that the strand bias in SHM may be caused by the preference of AID for the non-transcribed strand.

Transcription-dependent increase in multiple classes of base substitution mutations in Escherichia coli.

We showed previously that transcription in Escherichia coli promotes C. G-to-T. A transitions due to increased deamination of cytosines to uracils in the nontranscribed but not the transcribed strand (A. Beletskii and A. S. Bhagwat, Proc. Natl. Acad. Sci. USA 93:13919-13924, 1996). To study mutations other than that of C to T, we developed a new genetic assay that selects only base substitution mutations and additionally excludes C. G to T. A transitions. This novel genetic reversion system is based on mutations in a termination codon and involves positive selection for resistance to bleomycin or kanamycin. Using this genetic system, we show here that transcription from a strong promoter increases the level of non-C-to-T as well as C-to-T mutations. We find that high-level transcription increases the level of non-C-to-T mutations in DNA repair-proficient cells in three different sequence contexts in two genes and that the rate of mutation is higher by a factor of 2 to 4 under these conditions. These increases are not caused by a growth advantage for the revertants and are restricted to genes that are induced for transcription. In particular, high levels of transcription do not create a general mutator phenotype in E. coli. Sequence analysis of the revertants revealed that the frequency of several different base substitutions increased upon transcription of the bleomycin resistance gene and that G. C-to-T. A transversions dominated the spectrum in cells transcribing the gene. These results suggest that high levels of transcription promote many different spontaneous base substitutions in E. coli.