Expanded Systematic Evidence Map for Hundreds of Per- and Polyfluoroalkyl Substances (PFAS) and Comprehensive PFAS Human Health Dashboard.

BACKGROUND: Per- and polyfluoroalkyl substances (PFAS) encompass a class of chemically and structurally diverse compounds that are extensively used in industry and detected in the environment. The US Environmental Protection Agency (US EPA) 2021 PFAS Strategic Roadmap describes national research plans to address the challenge of PFAS. OBJECTIVES: Systematic Evidence Map (SEM) methods were used to survey and summarize available epidemiological and mammalian bioassay evidence that could inform human health hazard identification for a set of 345 PFAS that were identified by the US EPA's Center for Computational Toxicology and Exposure (CCTE) for in vitro toxicity and toxicokinetic assay testing and through interagency discussions on PFAS of interest. This work builds from the 2022 evidence map that collated evidence on a separate set of ∼150 PFAS. Like our previous work, this SEM does not include PFAS that are the subject of ongoing or completed assessments at the US EPA. METHODS: SEM methods were used to search, screen, and inventory mammalian bioassay and epidemiological literature from peer-reviewed and gray literature sources using manual review and machine-learning software. For each included study, study design details and health end points examined were summarized in interactive web-based literature inventories. Some included studies also underwent study evaluation and detailed extraction of health end point data. All underlying data is publicly available online as interactive visuals with downloadable metadata. RESULTS: More than 13,000 studies were identified from scientific databases. Screening processes identified 121 mammalian bioassay and 111 epidemiological studies that met screening criteria. Epidemiological evidence (available for 12 PFAS) mostly assessed the reproductive, endocrine, developmental, metabolic, cardiovascular, and immune systems. Mammalian bioassay evidence (available for 30 PFAS) commonly assessed effects in the reproductive, whole-body, nervous, and hepatic systems. Overall, 41 PFAS had evidence across mammalian bioassay and epidemiology data streams (roughly 11% of searched chemicals). DISCUSSION: No epidemiological and/or mammalian bioassay evidence were identified for most of the PFAS included in our search. Results from this SEM, our 2022 SEM on ∼150 PFAS, and other PFAS assessment products from the US EPA are compiled into a comprehensive PFAS dashboard that provides researchers and regulators an overview of the current PFAS human health landscape including data gaps and can serve as a scoping tool to facilitate prioritization of PFAS-related research and/or risk assessment activities. https://doi.org/10.1289/EHP13423.

Identifying xenobiotic metabolites with in silico prediction tools and LCMS suspect screening analysis.

Understanding the metabolic fate of a xenobiotic substance can help inform its potential health risks and allow for the identification of signature metabolites associated with exposure. The need to characterize metabolites of poorly studied or novel substances has shifted exposure studies towards non-targeted analysis (NTA), which often aims to profile many compounds within a sample using high-resolution liquid-chromatography mass-spectrometry (LCMS). Here we evaluate the suitability of suspect screening analysis (SSA) liquid-chromatography mass-spectrometry to inform xenobiotic chemical metabolism. Given a lack of knowledge of true metabolites for most chemicals, predictive tools were used to generate potential metabolites as suspect screening lists to guide the identification of selected xenobiotic substances and their associated metabolites. Thirty-three substances were selected to represent a diverse array of pharmaceutical, agrochemical, and industrial chemicals from Environmental Protection Agency's ToxCast chemical library. The compounds were incubated in a metabolically-active in vitro assay using primary hepatocytes and the resulting supernatant and lysate fractions were analyzed with high-resolution LCMS. Metabolites were simulated for each compound structure using software and then combined to serve as the suspect screening list. The exact masses of the predicted metabolites were then used to select LCMS features for fragmentation via tandem mass spectrometry (MS/MS). Of the starting chemicals, 12 were measured in at least one sample in either positive or negative ion mode and a subset of these were used to develop the analysis workflow. We implemented a screening level workflow for background subtraction and the incorporation of time-varying kinetics into the identification of likely metabolites. We used haloperidol as a case study to perform an in-depth analysis, which resulted in identifying five known metabolites and five molecular features that represent potential novel metabolites, two of which were assigned discrete structures based on in silico predictions. This workflow was applied to five additional test chemicals, and 15 molecular features were selected as either reported metabolites, predicted metabolites, or potential metabolites without a structural assignment. This study demonstrates that in some-but not all-cases, suspect screening analysis methods provide a means to rapidly identify and characterize metabolites of xenobiotic chemicals.

Advancing the science of a read-across framework for evaluation of data-poor chemicals incorporating systematic and new approach methods.

The assessment of human health hazards posed by chemicals traditionally relies on toxicity studies in experimental animals. However, most chemicals currently in commerce do not meet the minimum data requirements for hazard identification and dose-response analysis in human health risk assessment. Previously, we introduced a read-across framework designed to address data gaps for screening-level assessment of chemicals with insufficient in vivo toxicity information (Wang et al., 2012). It relies on inference by analogy from suitably tested source analogues to a target chemical, based on structural, toxicokinetic, and toxicodynamic similarity. This approach has been used for dose-response assessment of data-poor chemicals relevant to the U.S. EPA's Superfund program. We present herein, case studies of the application of this framework, highlighting specific examples of the use of biological similarity for chemical grouping and quantitative read-across. Based on practical knowledge and technological advances in the fields of read-across and predictive toxicology, we propose a revised framework. It includes important considerations for problem formulation, systematic review, target chemical analysis, analogue identification, analogue evaluation, and incorporation of new approach methods. This work emphasizes the integration of systematic methods and alternative toxicity testing data and tools in chemical risk assessment to inform regulatory decision-making.

Systematic evidence map (SEM) template: Report format and methods used for the US EPA Integrated Risk Information System (IRIS) program, Provisional Peer Reviewed Toxicity Value (PPRTV) program, and other "fit for purpose" literature-based human health analyses.

BACKGROUND: Systematic evidence maps (SEMs) are gaining visibility in environmental health for their utility to serve as problem formulation tools and assist in decision-making, especially for priority setting. SEMs are now routinely prepared as part of the assessment development process for the US Environmental Protection Agency (EPA) Integrated Risk Information System (IRIS) and Provisional Peer Reviewed Toxicity Value (PPRTV) assessments. SEMs can also be prepared to explore the available literature for an individual chemical or groups of chemicals of emerging interest. OBJECTIVES: This document describes the typical methods used to produce SEMs for the IRIS and PPRTV Programs, as well as "fit for purpose" applications using a variety of examples drawn from existing analyses. It is intended to serve as an example base template that can be adapted as needed for the specific SEM. The presented methods include workflows intended to facilitate rapid production. The Populations, Exposures, Comparators and Outcomes (PECO) criteria are typically kept broad to identify mammalian animal bioassay and epidemiological studies that could be informative for human hazard identification. In addition, a variety of supplemental content is tracked, e.g., studies presenting information on in vitro model systems, non-mammalian model systems, exposure-level-only studies in humans, pharmacokinetic models, and absorption, distribution, metabolism, and excretion (ADME). The availability of New Approach Methods (NAMs) evidence is also tracked (e.g., high throughput, transcriptomic, in silico, etc.). Genotoxicity studies may be considered as PECO relevant or supplemental material, depending on the topic and context of the review. Standard systematic review practices (e.g., two independent reviewers per record) and specialized software applications are used to search and screen the literature and may include the use of machine learning software. Mammalian bioassay and epidemiological studies that meet the PECO criteria after full-text review are briefly summarized using structured web-based extraction forms with respect to study design and health system(s) assessed. Extracted data is available in interactive visual formats and can be downloaded in open access formats. Methods for conducting study evaluation are also presented which is conducted on a case-by-case basis, depending on the usage of the SEM.

Use of systematic evidence maps within the US environmental protection agency (EPA) integrated risk information system (IRIS) program: Advancements to date and looking ahead.

Systematic evidence maps (SEMs) are increasingly used to inform decision-making and risk management priority-setting and to serve as problem formulation tools to refine the focus of questions that get addressed in full systematic reviews. Within the U.S. Environmental Protection Agency (EPA) Office of Research and Development (ORD) Integrated Risk Information System (IRIS), SEMs have been used to inform data gaps, determine the need for updated assessments, inform assessment priorities, and inform development of study evaluation considerations, among other uses. Increased utilization of SEMs across the environmental health field has the potential to increase transparency and efficiency for data gathering, problem formulation, read-across, and evidence surveillance. Use of the SEM templates published in the companion text (Thayer et al.) can promote harmonization in the environmental health community and create more opportunities for sharing extracted content.

Systematic Evidence Map for Over One Hundred and Fifty Per- and Polyfluoroalkyl Substances (PFAS).

BACKGROUND: Per- and polyfluoroalkyl substances (PFAS) are a large class of synthetic (man-made) chemicals widely used in consumer products and industrial processes. Thousands of distinct PFAS exist in commerce. The 2019 U.S. Environmental Protection Agency (U.S. EPA) Per- and Polyfluoroalkyl Substances (PFAS) Action Plan outlines a multiprogram national research plan to address the challenge of PFAS. One component of this strategy involves the use of systematic evidence map (SEM) approaches to characterize the evidence base for hundreds of PFAS. OBJECTIVE: SEM methods were used to summarize available epidemiological and animal bioassay evidence for a set of ∼150 PFAS that were prioritized in 2019 by the U.S. EPA's Center for Computational Toxicology and Exposure (CCTE) for in vitro toxicity and toxicokinetic assay testing. METHODS: Systematic review methods were used to identify and screen literature using manual review and machine-learning software. The Populations, Exposures, Comparators, and Outcomes (PECO) criteria were kept broad to identify mammalian animal bioassay and epidemiological studies that could inform human hazard identification. A variety of supplemental content was also tracked, including information on in vitro model systems; exposure measurement-only studies in humans; and absorption, distribution, metabolism, and excretion (ADME). Animal bioassay and epidemiology studies meeting PECO criteria were summarized with respect to study design, and health system(s) were assessed. Because animal bioassay studies with ≥21-d exposure duration (or reproductive/developmental study design) were most useful to CCTE analyses, these studies underwent study evaluation and detailed data extraction. All data extraction is publicly available online as interactive visuals with downloadable metadata. RESULTS: More than 40,000 studies were identified from scientific databases. Screening processes identified 44 animal and 148 epidemiology studies from the peer-reviewed literature and 95 animal and 50 epidemiology studies from gray literature that met PECO criteria. Epidemiological evidence (available for 15 PFAS) mostly assessed the reproductive, endocrine, developmental, metabolic, cardiovascular, and immune systems. Animal evidence (available for 40 PFAS) commonly assessed effects in the reproductive, developmental, urinary, immunological, and hepatic systems. Overall, 45 PFAS had evidence across animal and epidemiology data streams. DISCUSSION: Many of the ∼150 PFAS were data poor. Epidemiological and animal evidence were lacking for most of the PFAS included in our search. By disseminating this information, we hope to facilitate additional assessment work by providing the initial scoping literature survey and identifying key research needs. Future research on data-poor PFAS will help support a more complete understanding of the potential health effects from PFAS exposures. https://doi.org/10.1289/EHP10343.

Comparing the performance and coverage of selected in silico (liver) metabolism tools relative to reported studies in the literature to inform analogue selection in read-across: A case study.

Changes in the regulatory landscape of chemical safety assessment call for the use of New Approach Methodologies (NAMs) including read-across to fill data gaps. One critical aspect of analogue evaluation is the extent to which target and source analogues are metabolically similar. In this study, a set of 37 structurally diverse chemicals were compiled from the EPA ToxCast inventory to compare and contrast a selection of metabolism in silico tools, in terms of their coverage and performance relative to metabolism information reported in the literature. The aim was to build understanding of the scope and capabilities of these tools and how they could be utilised in a read-across assessment. The tools were Systematic Generation of Metabolites (SyGMa), Meteor Nexus, BioTransformer, Tissue Metabolism Simulator (TIMES), OECD Toolbox, and Chemical Transformation Simulator (CTS). Performance was characterised by sensitivity and precision determined by comparing predictions against literature reported metabolites (from 44 publications). A coverage score was derived to provide a relative quantitative comparison between the tools. Meteor, TIMES, Toolbox, and CTS predictions were run in batch mode, using default settings. SyGMa and BioTransformer were run with user-defined settings, (two passes of phase I and one pass of phase II). Hierarchical clustering revealed high similarity between TIMES and Toolbox. SyGMa had the highest coverage, matching an average of 38.63% of predictions generated by the other tools though was prone to significant overprediction. It generated 5,125 metabolites, which represented 54.67% of all predictions. Precision and sensitivity values ranged from 1.1-29% and 14.7-28.3% respectively. The Toolbox had the highest performance overall. A case study was presented for 3,4-Toluenediamine (3,4-TDA), assessed for the derivation of screening-level Provisional Peer Reviewed Toxicity Values (PPRTVs), was used to demonstrate the practical role in silico metabolism information can play in analogue evaluation as part of a read-across approach.

Clearly weighing the evidence in read-across can improve assessments of data-poor chemicals.

This paper provides a systematic weight-of-evidence method for read-across analyses of data-poor chemicals. The read-across technique extrapolates toxicity from analogous chemicals for which suitable test data are available to a target chemical. To determine that a candidate analogue is the 'best' and is sufficiently similar, the evidence for similarity of each candidate analogue to the target is weighed. We present a systematic weight of evidence method that provides transparency and imposes a consistent and rigorous inferential process. The method assembles relevant information concerning structure, physicochemical attributes, toxicokinetics, and toxicodynamics of the target and analogues. The information is then organized by evidence types and subtypes and weighted in terms of properties: relevance, strength, and reliability into weight levels, expressed as symbols. After evidence types are weighted, the bodies of evidence are weighted for collective properties: number, diversity, and coherence. Finally, the weights for the types and bodies of evidence are weighed for each analogue, and, if the overall weight of evidence is sufficient for one or more analogues, the analogue with the greatest weight is used to estimate the endpoint effect. We illustrate this WoE approach with a read-across analysis for screening the organochlorine contaminant, p,p'-dichlorodiphenyldichloroethane (DDD), for noncancer oral toxicity.

Exploring current read-across applications and needs among selected U.S. Federal Agencies.

Read-across is a well-established data gap-filling technique applied for regulatory purposes. In US Environmental Protection Agency's New Chemicals Program under TSCA, read-across has been used extensively for decades, however the extent of application and acceptance of read-across among U.S. federal agencies is less clear. In an effort to build read-across capacity, raise awareness of the state of the science, and work towards a harmonization of read-across approaches across U.S. agencies, a new read-across workgroup was established under the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). This is one of several ad hoc groups ICCVAM has convened to implement the ICCVAM Strategic Roadmap. In this article, we outline the charge and scope of the workgroup and summarize the current applications, tools used, and needs of the agencies represented on the workgroup for read-across. Of the agencies surveyed, the Environmental Protection Agency had the greatest experience in using read-across whereas other agencies indicated that they would benefit from gaining a perspective of the landscape of the tools and available guidance. Two practical case studies are also described to illustrate how the read-across approaches applied by two agencies vary on account of decision context.

A case study on the application of an expert-driven read-across approach in support of quantitative risk assessment of p,p'-dichlorodiphenyldichloroethane.

Deriving human health risk estimates for environmental chemicals has traditionally relied on in vivo toxicity databases to characterize potential adverse health effects and associated dose-response relationships. In the absence of in vivo toxicity information, new approach methods (NAMs) such as read-across have the potential to fill the required data gaps. This case study applied an expert-driven read-across approach to identify and evaluate analogues to fill non-cancer oral toxicity data gaps for p,p'-dichlorodiphenyldichloroethane (p,p'-DDD), an organochlorine contaminant known to occur at contaminated sites in the U.S. The source analogue p,p'-dichlorodiphenyltrichloroethane (DDT) and its no-observed-adverse-effect level of 0.05 mg/kg-day were proposed for the derivation of screening-level health reference values for the target chemical, p,p'-DDD. Among the primary similarity contexts (structure, toxicokinetics, and toxicodynamics), toxicokinetic considerations were instrumental in separating p,p'-DDT as the best source analogue from other potential candidates (p,p'-DDE and methoxychlor). In vitro high-throughput screening (HTS) assays from ToxCast were used to evaluate similarity in bioactivity profiles and make inferences toward plausible mechanisms of toxicity to build confidence in the read-across approach. This work demonstrated the value of NAMs such as read-across and in vitro HTS in human health risk assessment of environmental contaminants with the potential to inform regulatory decision-making.

Vesicular monoamine transporter 2 and the acute and long-term response to 3,4-(±)-methylenedioxymethamphetamine.

3,4-(±)-Methylenedioxymethamphetamine (MDMA, Ecstasy) is a ring-substituted amphetamine derivative with potent psychostimulant properties. The neuropharmacological effects of MDMA are biphasic in nature, initially causing synaptic monoamine release, primarily of serotonin (5-HT). Conversely, the long-term effects of MDMA manifest as prolonged depletions in 5-HT, and reductions in 5-HT reuptake transporter (SERT), indicative of serotonergic neurotoxicity. MDMA-induced 5-HT efflux relies upon disruption of vesicular monoamine storage, which increases cytosolic 5-HT concentrations available for release via a carrier-mediated mechanism. The vesicular monoamine transporter 2 (VMAT2) is responsible for packaging monoamine neurotransmitters into cytosolic vesicles. Thus, VMAT2 is a molecular target for a number of psychostimulant drugs, including methamphetamine and MDMA. We investigated the effects of depressed VMAT2 activity on the adverse responses to MDMA, via reversible inhibition of the VMAT2 protein with Ro4-1284. A single dose of MDMA (20 mg/kg, subcutaneous) induced significant hyperthermia in rats. Ro4-1284 (10 mg/kg, intraperitoneal) pretreatment prevented the thermogenic effects of MDMA, instead causing a transient decrease in body temperature. MDMA-treated rats exhibited marked increases in horizontal velocity and rearing behavior. In the presence of Ro4-1284, MDMA-mediated horizontal hyperlocomotion was delayed and attenuated, whereas rearing activity was abolished. Finally, Ro4-1284 prevented deficits in 5-HT content in rat cortex and striatum, and reduced depletions in striatal SERT staining, 7 days after MDMA administration. In summary, acute inhibition of VMAT2 by Ro4-1284 protected against MDMA-mediated hyperthermia, hyperactivity, and serotonergic neurotoxicity. The data suggest the involvement of VMAT2 in the thermoregulatory, behavioral, and neurotoxic effects of MDMA.

Catechol-o-methyltransferase and 3,4-({+/-})-methylenedioxymethamphetamine toxicity.

Metabolism of 3,4-(±)-methylenedioxymethamphetamine (MDMA) is necessary to elicit its neurotoxic effects. Perturbations in phase I and phase II hepatic enzymes can alter the neurotoxic profile of systemically administered MDMA. In particular, catechol-O-methyltransferase (COMT) plays a critical role in determining the fraction of MDMA that is converted to potentially neurotoxic metabolites. Thus, cytochrome P450 mediated demethylenation of MDMA, or its N-demethylated metabolite, 3,4-(±)-methylenedioxyamphetamine, give rise to the catechols, N-methyl-α-methyldopamine and α-methyldopamine, respectively. Methylation of these catechols by COMT limits their oxidation and conjugation to glutathione, a process that ultimately gives rise to neurotoxic metabolites. We therefore determined the effects of modulating COMT, a critical enzyme involved in determining the fraction of MDMA that is converted to potentially neurotoxic metabolites, on MDMA-induced toxicity. Pharmacological inhibition of COMT in the rat potentiated MDMA-induced serotonin deficits and exacerbated the acute MDMA-induced hyperthermic response. Using a genetic mouse model of COMT deficiency, in which mice lack a functional COMT gene, such mice displayed greater reductions in dopamine concentrations relative to their wild-type (WT) counterparts. Neither WT nor COMT deficient mice were susceptible to MDMA-induced decreases in serotonin concentrations. Interestingly, mice devoid of COMT were far more susceptible to the acute hyperthermic effects of MDMA, exhibiting greater increases in body temperature that ultimately resulted in death. Our findings support the view that COMT plays a pivotal role in determining the toxic response to MDMA.

Serotonin reuptake transporter deficiency modulates the acute thermoregulatory and locomotor activity response to 3,4-(±)-methylenedioxymethamphetamine, and attenuates depletions in serotonin levels in SERT-KO rats.

3,4-(±)-Methylenedioxymethamphetamine (MDMA) is a ring-substituted amphetamine derivative with potent psychostimulant properties. The neuropharmacological effects of MDMA are biphasic in nature, initially causing synaptic monoamine release, primarily of serotonin (5-HT), inducing thermogenesis and hyperactivity (5-HT syndrome). The long-term effects of MDMA manifest as a prolonged depletion in 5-HT, and structural damage to 5-HT nerve terminals. MDMA toxicity is in part mediated by an ability to inhibit the presynaptic 5-HT reuptake transporter (SERT). Using a SERT-knockout (SERT-KO) rat model, we determined the impact of SERT deficiency on thermoregulation, locomotor activity, and neurotoxicity in SERT-KO or Wistar-based wild-type (WT) rats exposed to MDMA. WT and SERT-KO animals exhibited the highest thermogenic responses to MDMA (four times 10 mg/kg, sc at 12 h intervals) during the diurnal (first and third) doses according to peak body temperature and area under the curve (∑°C × h) analysis. Although no differences in peak body temperature were observed between MDMA-treated WT and SERT-KO animals, ∑°C × h following the first MDMA dose was reduced in SERT-KO rats. Exposure to a single dose of MDMA stimulated horizontal velocity in both WT and SERT-KO rats, however, this effect was delayed and attenuated in the KO animals. Finally, SERT-KO rats were insensitive to MDMA-induced long-term (7 days) depletions in 5-HT and its metabolite, 5-hydroxyindole acetic acid, in both cortex and striatum. In conclusion, SERT deficiency modulated MDMA-mediated thermogenesis, hyperactivity and neurotoxicity in KO rats. The data confirm that the SERT is essential for the manifestation of the acute and long-term toxicities of MDMA.

Control of cell proliferation, endoreduplication, cell size, and cell death by the retinoblastoma-related pathway in maize endosperm.

The endosperm of cereal grains is one of the most valuable products of modern agriculture. Cereal endosperm development comprises different phases characterized by mitotic cell proliferation, endoreduplication, the accumulation of storage compounds, and programmed cell death. Although manipulation of these processes could maximize grain yield, how they are regulated and integrated is poorly understood. We show that the Retinoblastoma-related (RBR) pathway controls key aspects of endosperm development in maize. Down-regulation of RBR1 by RNAi resulted in up-regulation of RBR3-type genes, as well as the MINICHROMOSOME MAINTENANCE 2-7 gene family and PROLIFERATING CELL NUCLEAR ANTIGEN, which encode essential DNA replication factors. Both the mitotic and endoreduplication cell cycles were stimulated. Developing transgenic endosperm contained 42-58% more cells and ∼70% more DNA than wild type, whereas there was a reduction in cell and nuclear sizes. In addition, cell death was enhanced. The DNA content of mature endosperm increased 43% upon RBR1 down-regulation, whereas storage protein content and kernel weight were essentially not affected. Down-regulation of both RBR1 and CYCLIN DEPENDENT KINASE A (CDKA);1 indicated that CDKA;1 is epistatic to RBR1 and controls endoreduplication through an RBR1-dependent pathway. However, the repressive activity of RBR1 on downstream targets was independent from CDKA;1, suggesting diversification of RBR1 activities. Furthermore, RBR1 negatively regulated CDK activity, suggesting the presence of a feedback loop. These results indicate that the RBR1 pathway plays a major role in regulation of different processes during maize endosperm development and suggest the presence of tissue/organ-level regulation of endosperm/seed homeostasis.

Positive regulation of minichromosome maintenance gene expression, DNA replication, and cell transformation by a plant retinoblastoma gene.

Retinoblastoma-related (RBR) genes inhibit the cell cycle primarily by repressing adenovirus E2 promoter binding factor (E2F) transcription factors, which drive the expression of numerous genes required for DNA synthesis and cell cycle progression. The RBR-E2F pathway is conserved in plants, but cereals such as maize are characterized by having a complex RBR gene family with at least 2 functionally distinct members, RBR1 and RBR3. Although RBR1 has a clear cell cycle inhibitory function, it is not known whether RBR3 has a positive or negative role. By uncoupling RBR3 from the negative regulation of RBR1 in cultured maize embryos through a combination of approaches, we demonstrate that RBR3 has a positive and critical role in the expression of E2F targets required for the initiation of DNA synthesis, DNA replication, and the efficiency with which transformed plants can be obtained. Titration of endogenous RBR3 activity through expression of a dominant-negative allele with a compromised pocket domain suggests that these RBR3 functions require an activity distinct from its pocket domain. Our results indicate a cell cycle pathway in maize, in which 2 RBR genes have specific and opposing functions. Thus, the paradigm that RBR genes are negative cell cycle regulators cannot be considered universal.