Updated systematic assessment of human, animal and mechanistic evidence demonstrates lack of human carcinogenicity with consumption of aspartame.

Aspartame has been studied extensively and evaluated for its safety in foods and beverages yet concerns for its potential carcinogenicity have persisted, driven primarily by animal studies conducted at the Ramazzini Institute (RI). To address this controversy, an updated systematic review of available human, animal, and mechanistic data was conducted leveraging critical assessment tools to consider the quality and reliability of data. The evidence base includes 12 animal studies and >40 epidemiological studies reviewed by the World Health Organization which collectively demonstrate a lack of carcinogenic effect. Assessment of >1360 mechanistic endpoints, including many guideline-based genotoxicity studies, demonstrate a lack of activity associated with endpoints grouped to key characteristics of carcinogens. Other non-specific mechanistic data (e.g., mixed findings of oxidative stress across study models, tissues, and species) do not provide evidence of a biologically plausible carcinogenic pathway associated with aspartame. Taken together, available evidence supports that aspartame consumption is not carcinogenic in humans and that the inconsistent findings of the RI studies may be explained by flaws in study design and conduct (despite additional analyses to address study limitations), as acknowledged by authoritative bodies.

Assessment of the applicability of the threshold of toxicological concern for per- and polyfluoroalkyl substances.

While toxicity information is available for selected PFAS, little or no information is available for most, thereby necessitating a resource-effective approach to screen and prioritize those needing further safety assessment. The threshold of toxicological concern (TTC) approach proposes a de minimis exposure value based on chemical structure and toxicology of similar substances. The applicability of the TTC approach to PFAS was tested by incorporating a data set of no-observed-adverse-effect level (NOAEL) values for 27 PFAS into the Munro TTC data set. All substances were assigned into Cramer Class III and the cumulative distribution of the NOAELs evaluated. The TTC value for the PFAS-enriched data set was not statistically different compared to the Munro data set. Derived human exposure level for the PFAS-enriched data set was 1.3 µg/kg/day. Structural chemical profiles showed the PFAS-enriched data set had distinct chemotypes with lack of similarity to substances in the Munro data set using Maximum Common Structures. The incorporation of these 27 PFAS did not significantly change TTC Cramer Class III distribution and expanded the chemical space, supporting the potential use of the TTC approach for PFAS chemicals.

Overall lack of genotoxic activity among five common low- and no-calorie sweeteners: A contemporary review of the collective evidence.

Low- and no-calorie sweeteners (LNCS) are food additives that have been widely consumed for many decades. Their safety has been well established by authoritative bodies globally and is re-evaluated periodically. The objective herein was to survey and summarize the genotoxicity potential of five commonly utilized LNCS: acesulfame potassium (Ace-K), aspartame, saccharin, steviol glycosides and sucralose. Data from peer-reviewed literature and the ToxCast/Tox21 database were evaluated and integrated with the most recent weight-of-evidence evaluations from authoritative sources. Emphasis was placed on assays most frequently considered for hazard identification and risk assessment: mutation, clastogenicity and/or aneugenicity, and indirect DNA damage, such as changes in DNA repair mechanisms or gene expression data. These five sweeteners have been collectively evaluated in hundreds of in vivo or in vitro studies that employ numerous testing models, many of which have been conducted according to specific testing guidelines. The weight-of-evidence demonstrates overall negative findings across assay types for each sweetener when considering the totality of study design, reliability and reporting quality, as well as the lack of carcinogenic responses (or lack of responses relevant to humans) in animal cancer bioassays as well as observational studies in humans. This conclusion is consistent with the opinions of authoritative sources that have consistently determined that these sweeteners lack mutagenic and genotoxic potential.

CEBS: a comprehensive annotated database of toxicological data.

The Chemical Effects in Biological Systems database (CEBS) is a comprehensive and unique toxicology resource that compiles individual and summary animal data from the National Toxicology Program (NTP) testing program and other depositors into a single electronic repository. CEBS has undergone significant updates in recent years and currently contains over 11 000 test articles (exposure agents) and over 8000 studies including all available NTP carcinogenicity, short-term toxicity and genetic toxicity studies. Study data provided to CEBS are manually curated, accessioned and subject to quality assurance review prior to release to ensure high quality. The CEBS database has two main components: data collection and data delivery. To accommodate the breadth of data produced by NTP, the CEBS data collection component is an integrated relational design that allows the flexibility to capture any type of electronic data (to date). The data delivery component of the database comprises a series of dedicated user interface tables containing pre-processed data that support each component of the user interface. The user interface has been updated to include a series of nine Guided Search tools that allow access to NTP summary and conclusion data and larger non-NTP datasets. The CEBS database can be accessed online at http://www.niehs.nih.gov/research/resources/databases/cebs/.

Mutation spectra of Kras and Tp53 in urethral and lung neoplasms in B6C3F1 mice treated with 3,3',4,4'-tetrachloroazobenzene.

3,3',4,4'-tetrachloroazobenzene (TCAB) is a contaminant formed during manufacture of various herbicide compounds. A recent National Toxicology Program study showed B6C3F1 mice exposed to TCAB developed a treatment-related increase in lung carcinomas in the high-dose group, and urethral carcinomas, an extremely rare lesion in rodents, in all dose groups. As the potential for environmental exposure to TCAB is widespread, and the mechanisms of urethral carcinogenesis are unknown, TCAB-induced urethral and pulmonary tumors were evaluated for alterations in critical human cancer genes, Kras and Tp53. Uroplakin III, CK20, and CK7 immunohistochemistry was performed to confirm the urothelial origin of urethral tumors. TCAB-induced urethral carcinomas harbored transforming point mutations in K-ras (38%) and Tp53 (63%), and 71% displayed nuclear TP53 expression, consistent with formation of mutant protein. Transition mutations accounted for 88% of Tp53 mutations in urethral carcinomas, suggesting that TCAB or its metabolites target guanine or cytosine bases and that these mutations are involved in urethral carcinogenesis. Pulmonary carcinomas in TCAB-exposed animals harbored similar rates of Tp53 (55%) and Kras (36%) mutations as urethral carcinomas, suggesting that TCAB may induce mutations at multiple sites by a common mechanism. In conclusion, TCAB is carcinogenic at multiple sites in male and female B6C3F1 mice through mechanisms involving Tp53 and Kras mutation.

Genetic pathways to colorectal cancer.

The colorectal cancer paradigm explains how genetic and histological changes lead normal epithelial cell to transform into pre-malignant adenomas then progress to malignant carcinomas. Using the Genetic Alterations in Cancer Knowledge System intragenic allele loss and gene mutation data from approximately 9000 colorectal tumors were compared to the model of colorectal tumor development. The distribution of mutations along the TP53 codons as a function of tumorigenesis also was analyzed. Alterations of APC, KRAS and TP53 were observed in a higher percentage of adenocarcinomas compared to adenomas (P<0.05) indicating that the alterations accumulated with malignancy. Alterations in BRAF, CTNNB, HRAS and NRAS were infrequent regardless of morphology. Differences were observed in the distribution of TP53 mutations with tumorigenesis. Mutations (single base substitutions) occurred most frequently at codons 175 and 273 in both tumor types; however, in adenocarcinomas the mutation incidence at codon 248 was approximately three times that reported in adenomas. It is proposed that the higher incidence of mutation at codon 248 is a later event in colorectal tumorigenesis that occurs as the tumors become malignant.

Association of sperm protein 17 with A-kinase anchoring protein 3 in flagella.

BACKGROUND: Sperm protein 17 (Sp17) is a three-domain protein that contains: 1) a highly conserved N-terminal domain that is 45% identical to the human type II alpha regulatory subunit (RII alpha) of protein kinase A (PKA); 2) a central sulphated carbohydrate-binding domain; and 3) a C-terminal Ca++/calmodulin (CaM) binding domain. Although Sp17 was originally discovered and characterized in spermatozoa, its mRNA has now been found in a variety of normal mouse and human tissues. However, Sp17 protein is found predominantly in spermatozoa, cilia and human neoplastic cell lines. This study demonstrates that Sp17 from spermatozoa binds A-kinase anchoring protein 3 (AKAP3), confirming the functionality of the N-terminal domain. METHODS: In this study in vitro precipitation and immunolocalization demonstrate that Sp17 binds to AKAP3 (AKAP110) in spermatozoa. RESULTS: Sp17 is present in the head and tail of spermatozoa, in the tail it is in the fibrous sheath, which contains AKAP3 and AKAP4. Recombinant AKAP3 and AKAP4 RII binding domains were synthesized as glutathione S-transferase (GST) fusion proteins immobilized on glutathione-agarose resin and added to CHAPS extracts of human spermatozoa. Western blots of bound and eluted proteins probed with anti-Sp17 revealed that AKAP3 bound and precipitated a significant level of Sp17 while AKAP4 did not. AKAP4 binds AKAP3 and expression of AKAP3 is reduced in AKAP4 knockout sperm, therefore we tested AKAP4 knockout spermatozoa for Sp17 and found that there was a reduction in the amount of Sp17 expressed when compared to wild type spermatozoa. Co-localization of AKAP3 and Sp17 by immunofluorescence was demonstrated along the length of the principal piece of the flagella. CONCLUSIONS: As predicted by its N-terminal domain that is 45% identical to the human RIIalpha of PKA, Sp17 from spermatozoa binds the RII binding domain of AKAP3 along the length of the flagella.

Analysis of recombinant mouse zona pellucida protein 2 (ZP2) constructs for immunocontraception.

In this study we have examined the potential of recombinant mouse zona pellucida glycoprotein 2 (ZP2) as a target for immunocontraception. Immunogenicity studies and fertility trials were performed in outbred Swiss-Webster mice using four ZP2 constructs: Val(35)-Gly(200) (ZP2(V35-G200)), Val(35)-Leu(331) (ZP2(V35-L331)), Pro(325)-Ala(637) (ZP2(P325-A637)), and Val(35)-Ala(637) (ZP2(V35-A637)). A significant antibody response occurred to three of the four immunogens, however antibodies capable of recognizing native ZP occurred only after immunization with ZP2(V35-A637) and ZP2(P325-A637). Only immunization with ZP2(V35-A637) correlated with a reduction in fertility. Examination of the physiological basis for infertility revealed that: (1) passive transfer of ZP2 antiserum induced infertility in non-immune mice; (2) ovaries of infertile mice appeared histologically normal; (3) infertile mice produced normal numbers of eggs and (4) ZP of ovulated eggs from infertile mice demonstrated a significant reduction in the number of sperm bound compared to eggs from adjuvant controls. Infertility can be caused entirely by ZP2 antibodies without the incidence of significant ovarian pathology. This study also demonstrated that immunization with the bioactive (sperm binding) region of ZP2, recombinant ZP2(V35-G200), did not result in a significant immune response that recognized native ZP or inhibited fertility. Consequently we designed a ZP2-sperm antigen construct, replacing the C-terminal region of ZP2 with Sp17. This construct proved to be immunogenic and reduce fertility while directing the immune response to the Val(35)-Gly(200) region of ZP2.