Resistance-guided mining of bacterial genotoxins defines a family of DNA glycosylases

Unique DNA repair enzymes that provide self-resistance against genotoxic natural products have been discovered recently in bacterial biosynthetic gene clusters (BGCs). The DNA glycosylase AlkZ belongs to a superfamily of uncharacterized proteins found in antibiotic producers and pathogens, but despite its importance to azinomycin B resistance, the roles of AlkZ orthologs in production of other natural products are unknown. Here, we analyze the genomic distribution and use a resistance-based genome mining approach to identify Streptomyces AlkZ homologs associated with known and uncharacterized BGCs. We show that the ortholog associated with synthesis of the alkylating agent hedamycin excises hedamycin-DNA adducts and provides resistance to the genotoxin in cells. Our results define AlkZ in self-resistance to specific antimicrobials and implicate a related but distinct homolog, which we name AlkX, in protection against an array of genotoxins. This work provides a framework for targeted discovery of new genotoxic compounds with therapeutic potential.

Alkenylbenzenes in Foods: Aspects Impeding the Evaluation of Adverse Health Effects

Alkenylbenzenes are naturally occurring secondary plant metabolites, primarily present in different herbs and spices, such as basil or fennel seeds. Thus, alkenylbenzenes, such as safrole, methyleugenol, and estragole, can be found in different foods, whenever these herbs and spices (or extracts thereof) are used for food production. In particular, essential oils or other food products derived from the aforementioned herbs and spices, such as basil-containing pesto or plant food supplements, are often characterized by a high content of alkenylbenzenes. While safrole or methyleugenol are known to be genotoxic and carcinogenic, the toxicological relevance of other alkenylbenzenes (e.g., apiol) regarding human health remains widely unclear. In this review, we will briefly summarize and discuss the current knowledge and the uncertainties impeding a conclusive evaluation of adverse effects to human health possibly resulting from consumption of foods containing alkenylbenzenes, especially focusing on the genotoxic compounds, safrole, methyleugenol, and estragole.

Threshold of Toxicological Concern—An Update for Non-Genotoxic Carcinogens

The Threshold of Toxicological Concern (TTC) concept can be applied to organic compounds with the known chemical structure to derive a threshold for exposure, below which a toxic effect on human health by the compound is not expected. The TTC concept distinguishes between carcinogens that may act as genotoxic and non-genotoxic compounds. A positive prediction of a genotoxic mode of action, either by structural alerts or experimental data, leads to the application of the threshold value for genotoxic compounds. Non-genotoxic substances are assigned to the TTC value of their respective Cramer class, even though it is recognized that they could test positive in a rodent cancer bioassay. This study investigated the applicability of the Cramer classes specifically to provide adequate protection for non-genotoxic carcinogens. For this purpose, benchmark dose levels based on tumor incidence were compared with no observed effect levels (NOELs) derived from non-, pre- or neoplastic lesions. One key aspect was the categorization of compounds as non-genotoxic carcinogens. The recently finished CEFIC LRI project B18 classified the carcinogens of the Carcinogenicity Potency DataBase (CPDB) as either non-genotoxic or genotoxic compounds based on experimental or in silico data. A detailed consistency check resulted in a dataset of 137 non-genotoxic organic compounds. For these 137 compounds, NOEL values were derived from high quality animal studies with oral exposure and chronic duration using well-known repositories, such as RepDose, ToxRef, and COSMOS DB. Further, an effective tumor dose (ETD10) was calculated and compared with the lower confidence limit on benchmark dose levels (BMDL10) derived by model averaging. Comparative analysis of NOEL/EDT10/BMDL10 values showed that potentially bioaccumulative compounds in humans, as well as steroids, which both belong to the exclusion categories, occur predominantly in the region of the fifth percentiles of the distributions. Excluding these 25 compounds resulted in significantly higher but comparable fifth percentile chronic NOEL and BMDL10 values, while the fifth percentile EDT10 value was slightly higher but not statistically significant. The comparison of the obtained distributions of NOELs with the existing Cramer classes and their derived TTC values supports the application of Cramer class thresholds to all non-genotoxic compounds, such as non-genotoxic carcinogens.

In vivo comet assay in rabbit corneal epithelial cells following ocular instillation with genotoxic compounds

Background The in vivo comet assay is used to evaluate the genotoxic potential of compounds by detecting DNA strand breaks in cells isolated from animal tissue. The comet assay of hepatocytes is well established; however, the levels of systemic drug exposure following systemic administration are often insufficient to evaluate the genotoxic potential of compounds on the ocular surface following ocular instillation. To investigate the possibility of using the comet assay as a genotoxic evaluation tool for the ocular surface, we performed this assay on the corneal epithelial cells of rabbit eyes 2 h after the single ocular instillation of five genotoxic compounds, namely ethidium bromide, 1,1′-dimethyl-4,4′-bipyridinium dichloride (paraquat), methyl methanesulfonate (MMS), acrylamide, and 4-nitroquinoline 1-oxide (4-NQO). Results The mean % tail DNA, as an indicator of DNA damage, in the corneal epithelial cells treated with ethidium bromide, MMS, and 4-NQO exhibited statistically significant increases compared with those in the negative controls (saline or 5 % dimethyl sulfoxide in saline). However, paraquat and acrylamide did not increase the mean % tail DNA, presumably because of the high antioxidant levels and low cytochrome P450 levels present in the corneal epithelium, respectively. Conclusions The comet assay was able to detect genotoxic potential on the ocular surface following ocular instillation with genotoxic compounds. The study findings indicate that the in vivo comet assay may provide a useful tool for assessing the genotoxicity of compounds topically administrated on the ocular surface under mimicking clinical condition.

Genotoxicity of Nanomaterials: Advanced In Vitro Models and High Throughput Methods for Human Hazard Assessment—A Review

Changes in the genetic material can lead to serious human health defects, as mutations in somatic cells may cause cancer and can contribute to other chronic diseases. Genotoxic events can appear at both the DNA, chromosomal or (during mitosis) whole genome level. The study of mechanisms leading to genotoxicity is crucially important, as well as the detection of potentially genotoxic compounds. We consider the current state of the art and describe here the main endpoints applied in standard human in vitro models as well as new advanced 3D models that are closer to the in vivo situation. We performed a literature review of in vitro studies published from 2000–2020 (August) dedicated to the genotoxicity of nanomaterials (NMs) in new models. Methods suitable for detection of genotoxicity of NMs will be presented with a focus on advances in miniaturization, organ-on-a-chip and high throughput methods.

Aneugen Versus Clastogen Evaluation and Oxidative Stress-Related Mode-of-Action Assessment of Genotoxic Compounds Using the ToxTracker Reporter Assay

Understanding the mode-of-action (MOA) of genotoxic compounds and differentiating between direct DNA interaction and indirect genotoxicity is crucial for their reliable safety assessment. ToxTracker is a stem cell-based reporter assay that detects activation of various cellular responses that are associated with genotoxicity and cancer. ToxTracker consists of 6 different GFP reporter cell lines that can detect the induction of DNA damage, oxidative stress, and protein damage in a single test. The assay can thereby provide insight into the MOA of compounds. Genotoxicity is detected in ToxTracker by activation of 2 independent GFP reporters. Activation of the Bscl2-GFP reporter is associated with induction of DNA adducts and subsequent inhibition of DNA replication and the Rtkn-GFP reporter is activated following the formation of DNA double-strand breaks. Here, we show that the differential activation of these 2 genotoxicity reporters could be used to further differentiate between a DNA reactive and clastogenic or a non-DNA-reactive aneugenic MOA of genotoxic compounds. For further classification of aneugenic and clastogenic compounds, the ToxTracker assay was extended with cell cycle analysis and aneuploidy assessment. The extension was validated using a selection of 16 (genotoxic) compounds with a well-established MOA. Furthermore, indirect genotoxicity related to the production of reactive oxygen species was investigated using the DNA damage and oxidative stress ToxTracker reporters in combination with different reactive oxygen species scavengers. With these new extensions, ToxTracker was able to accurately classify compounds as genotoxic or nongenotoxic and could discriminate between DNA-reactive compounds, aneugens, and indirect genotoxicity caused by oxidative stress.

Changes in 3-, 2-Monochloropropandiol and Glycidyl Esters during a Conventional Baking System with Addition of Antioxidants

Shortening derived from palm oil is widely used in baking applications. However, palm oil and the related products are reported to contain high levels of monochloropropandiol (MCPD) ester and glycidyl ester (GE). MCPD and glycidol are known as process contaminants, which are carcinogenic and genotoxic compounds, respectively. The objective was to evaluate the effects of antioxidant addition in palm olein and stearin to the content of MCPD esters and GE in baked cake. Butylated hydroxyanisole (BHA), rosemary extract and tocopherol were used to fortify the samples at 200 mg/kg and in combinations (400, 600 and 800 mg/kg rosemary or tocopherol combined with 200 mg/kg BHA). The MCPD esters and GE content, radical formation and the quality of the fats portion were analyzed. The results showed that palm olein fortified with rosemary extract yielded less 2-MCPD ester. The GE content was lower when soft stearin was fortified with rosemary. ESR spectrometry measurements showed that the antioxidants were effective to reduce radical formation. The synergistic effects of combining antioxidants controlled the contaminants formation. In conclusion, oxidation stability was comparable either in the single or combined antioxidants. Tocopherol in combination with BHA was more effective in controlling the MCPD esters and GE formation.

Probiotic antigenotoxic activity as a DNA bioprotective tool: a minireview with focus on endocrine disruptors

ABSTRACT Nowadays, the interest in the role of dietary components able to influence the composition and the activity of the intestinal microbiota and, consequently, to modulate the risk of genotoxicity and colon cancer is increasing in the scientific community. Within this topic, the microbial ability to have a protective role at gastrointestinal level by counteracting the biological activity of genotoxic compounds, and thus preventing the DNA damage, is deemed important in reducing gut pathologies and is considered a new tool for probiotics and functional foods. A variety of genotoxic compounds can be found in the gut and, besides food-related mutagens and other DNA-reacting compounds, there is a group of pollutants commonly used in food packaging and/or in thousands of everyday products called endocrine disruptors (EDs). EDs are exogenous substances that alter the functions of the endocrine system through estrogenic and anti-estrogenic activity, which interfere with normal hormonal function in human and wildlife. Thus, this paper summarizes the main applications of probiotics, mainly lactobacilli, as a bio-protective tool to counteract genotoxic and mutagenic agents, by biologically inhibiting the related DNA damage in the gut and highlights the emerging perspectives to enlarge and further investigate the microbial bio-protective role at intestinal level.