Combined in vitro and in silico mechanistic approach to explore the potential of Alternaria mycotoxins alternariol and altertoxin II to hamper γH2AX formation in DNA damage signaling pathways.

Risk assessment of food and environmental contaminants is faced by substantial data gaps and novel strategies are needed to support science-based regulatory actions. The Alternaria mycotoxins alternariol (AOH) and altertoxin II (ATXII) have garnered attention for their possible genotoxic effects. Nevertheless, data currently available are rather scattered, hindering a comprehensive hazard characterization. This study combined in vitro/in silico approaches to elucidate the potential of AOH and ATXII to induce double-strand breaks (DSBs) in HepG2 cells. Furthermore, it examines the impact of co-exposure to AOH and the DSB-inducing drug doxorubicin (Doxo) on γH2AX expression. AOH slightly increased γH2AX expression, whereas ATXII did not elicit this response. Interestingly, AOH suppressed Doxo-induced γH2AX expression, despite evidence of increased DNA damage in the comet assay. Building on these observations, AOH was postulated to inhibit γH2AX-forming kinases. Along this line, in silico analysis supported AOH potential interaction with the ATP-binding sites of these kinases and immunofluorescence experiments showed decreased intracellular phosphorylation events. Similarly, in silico results suggested that ATXII might also interact with these kinases. This study emphasizes the importance of understanding the implications of AOH-induced γH2AX expression inhibition on DNA repair processes and underscores the need for caution when interpreting γH2AX assay results.

Hazard characterization of Alternaria toxins to identify data gaps and improve risk assessment for human health.

Fungi of the genus Alternaria are ubiquitous plant pathogens and saprophytes which are able to grow under varying temperature and moisture conditions as well as on a large range of substrates. A spectrum of structurally diverse secondary metabolites with toxic potential has been identified, but occurrence and relative proportion of the different metabolites in complex mixtures depend on strain, substrate, and growth conditions. This review compiles the available knowledge on hazard identification and characterization of Alternaria toxins. Alternariol (AOH), its monomethylether AME and the perylene quinones altertoxin I (ATX-I), ATX-II, ATX-III, alterperylenol (ALP), and stemphyltoxin III (STTX-III) showed in vitro genotoxic and mutagenic properties. Of all identified Alternaria toxins, the epoxide-bearing analogs ATX-II, ATX-III, and STTX-III show the highest cytotoxic, genotoxic, and mutagenic potential in vitro. Under hormone-sensitive conditions, AOH and AME act as moderate xenoestrogens, but in silico modeling predicts further Alternaria toxins as potential estrogenic factors. Recent studies indicate also an immunosuppressive role of AOH and ATX-II; however, no data are available for the majority of Alternaria toxins. Overall, hazard characterization of Alternaria toxins focused, so far, primarily on the commercially available dibenzo-α-pyrones AOH and AME and tenuazonic acid (TeA). Limited data sets are available for altersetin (ALS), altenuene (ALT), and tentoxin (TEN). The occurrence and toxicological relevance of perylene quinone-based Alternaria toxins still remain to be fully elucidated. We identified data gaps on hazard identification and characterization crucial to improve risk assessment of Alternaria mycotoxins for consumers and occupationally exposed workers.

Genotoxic and Mutagenic Effects of the Alternaria Mycotoxin Alternariol in Combination with the Process Contaminant Acrylamide.

Humans are constantly exposed to mixtures of different xenobiotics through their diet. One emerging concern is the Alternaria mycotoxin alternariol (AOH), which can occur in foods typically contaminated by the process contaminant acrylamide (AA). AA is a byproduct of the Maillard reaction produced in carbohydrate-rich foods during thermal processing. Given the genotoxic properties of AOH and AA as single compounds, as well as their potential co-occurrence in food, this study aimed to assess the cytotoxic, genotoxic, and mutagenic effects of these compounds in combination. Genotoxicity was assessed in HepG2 cells by quantifying the phosphorylation of the histone γ-H2AX, induced as a response to DNA double-strand breaks (DSBs). Mutagenicity was tested in Salmonella typhimurium strains TA98 and TA100 by applying the Ames microplate format test. Our results showed the ability of AOH and AA to induce DSBs and increase revertant numbers in S. typhimurium TA100, with AOH being more potent than AA. However, no synergistic effects were observed during the combined treatments. Notably, the results of the study suggest that the compounds exert mutagenic effects primarily through base pair substitutions. In summary, the data indicate no immediate cause for concern regarding synergistic health risks associated with the consumption of foods co-contaminated with AOH and AA.

A review on the ecotoxicity of macrocyclic lactones and benzimidazoles on aquatic organisms.

Despite its wide production and several applications, veterinary antiparasitics from macrocyclic lactones and benzimidazole classes have not received much scientific attention concerning their environmental risks. Thus, we aimed to provide insights into the state of the environmental research on macrocyclic lactone and benzimidazole parasiticides, emphasizing their toxicity to non-target aquatic organisms. We searched for relevant information on these pharmaceutical classes on PubMed and Web of Science. Our search yielded a total of 45 research articles. Most articles corresponded to toxicity testing (n = 29), followed by environmental fate (n = 14) and other issues (n = 2) of selected parasiticides. Macrocyclic lactones were the most studied chemical group (65% of studies). Studies were conducted mainly with invertebrate taxa (70%), with crustaceans being the most predominant group (n = 27; 51%). Daphnia magna was the most used species (n = 8; 15%). Besides, it also proved to be the most sensitive organism, yielding the lowest toxicity measure (EC50 0.25 µg/L for decreased mobility after 48 h-abamectin exposure) reported. Moreover, most studies were performed in laboratory settings, tracking a limited number of endpoints (acute mortality, immobility, and community disturbance). We posit that macrocyclic lactones and benzimidazoles warrant coordinated action to understand their environmental risks.

Cytotoxicities of Co-occurring alternariol, alternariol monomethyl ether and tenuazonic acid on human gastric epithelial cells.

Alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TeA) are the three major Alternaria toxin contaminants in food. In the present study, we conducted their single and combined toxicity analyses using human gastric epithelial cell line (GES-1) that was first exposed to the toxins when they entered the human body. By comparing the cytotoxicity IC50, we found that compared to several other mycotoxins with limit standards there was cytotoxicity DON > OTA > AME > AOH > ZEN > TeA. Further, we obtained combination index (CI)-isobologram equation by the Chou-Talalay method according to a toxin ratio of 1:1:2 and carried out the combined toxicity analysis of the three binary and ternary compounds, and the results showed that AOH + AME + TeA showed synergistic toxic effects. Based on the co-occurring status, we also carried out the combined toxicity analysis of AME and AOH at different ratios and found antagonistic effects at low cytotoxic concentrations as well as synergistic and additive effects at high concentrations. Also, we found that all three and their combinations caused apoptosis, activation of caspase-3 cleavage, activation of DNA damage pathways ATR-Chk1-P53 and ATM-Chk2-P53. In conclusion, we used GES-1 cells to inform the risk of coaction of AOH, AME, and TeA in dietary exposure.

Encapsulation of Cynara Cardunculus Guaiane-type Lactones in Fully Organic Nanotubes Enhances Their Phytotoxic Properties.

The encapsulation of bioactive natural products has emerged as a relevant tool for modifying the poor physicochemical properties often exhibited by agrochemicals. In this regard, natural guaiane-type sesquiterpene lactones isolated from Cynara cardunculus L. have been encapsulated in a core/shell nanotube@agrochemical system. Monitoring of the F and O signals in marked sesquiterpenes confirmed that the compound is present in the nanotube cavity. These structures were characterized using scanning transmission electron microscopy-X-ray energy-dispersive spectrometry techniques, which revealed the spatial layout relationship and confirmed encapsulation of the sesquiterpene lactone derivative. In addition, biological studies were performed with aguerin B (1), cynaropicrin (2), and grosheimin (3) on the inhibition of germination, roots, and shoots in weeds (Phalaris arundinacea L., Lolium perenne L., and Portulaca oleracea L.). Encapsulation of lactones in nanotubes gives better results than those for the nonencapsulated compounds, thereby reinforcing the application of fully organic nanotubes for the sustainable use of agrochemicals in the future.

RIFM fragrance ingredient safety assessment, 5-hydroxy-7-decenoic acid δ-lactone, CAS Registry Number 25524-95-2.

The existing information supports the use of this material as described in this safety assessment. 5-Hydroxy-7-decenoic acid δ-lactone was evaluated for genotoxicity, repeated dose toxicity, reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization, and environmental safety. Data from read-across material tetrahydro-6-(3-pentenyl)-2H-pyran-2-one (CAS # 32764-98-0) show that 5-hydroxy-7-decenoic acid δ-lactone is not expected to be genotoxic. The repeated dose, reproductive, and local respiratory toxicity endpoints were evaluated using the Threshold of Toxicological Concern (TTC) for a Cramer Class I material, and the exposure to 5-hydroxy-7-decenoic acid δ-lactone is below the TTC (0.03 mg/kg/day, 0.03 mg/kg/day, and 1.4 mg/day, respectively). Data show that there are no safety concerns for 5-hydroxy-7-decenoic acid δ-lactone for skin sensitization under the current declared levels of use. The phototoxicity/photoallergenicity endpoints were evaluated based on ultraviolet/visible (UV/Vis) spectra; 5-hydroxy-7-decenoic acid δ-lactone is not expected to be phototoxic/photoallergenic. The environmental endpoints were evaluated; 5-Hydroxy-7-decenoic acid δ-lactone was found not to be Persistent, Bioaccumulative, and Toxic (PBT) as per the International Fragrance Association (IFRA) Environmental Standards, and its risk quotients, based on its current volume of use in Europe and North America (i.e., Predicted Environmental Concentration/Predicted No Effect Concentration [PEC/PNEC]), are <1.

Alternariol monomethyl ether toxicity and genotoxicity in male Sprague-Dawley rats: 28-Day in vivo multi-endpoint assessment.

Alternariol monomethyl ether (AME), a typical Alternaria toxin, has often been detected in grains. We have measured the general toxicity and genotoxicity of AME with a 28-day multi-endpoint (Pig-a assay + in vivo micronucleus [MN] test + comet assay) platform. Male Sprague-Dawley rats were administered AME (1.84, 3.67, or 7.35 µg/kg body weight/day), N-Ethyl-N-nitrosourea (40 mg/kg body weight/day), or corn oil by gavage for 28 consecutive days. Another group (AME-high-dose + recovery) was maintained for a further 14 days after the end of the AME administration. Hematology and serum biochemistry results suggested that AME might compromise the immune system. The histopathology results indicated that AME can cause liver (inflammatory cell infiltration, steatosis, and edema), kidney (renal glomerular atrophy), and spleen (white pulp atrophy) damage. The genotoxicity results showed that AME can induce gene mutations, chromosome breakage, and DNA damage, but the effects were diminished after the recovery period. According to point-of-departure analysis (BMDL10), the risk to the population of exposure to AME cannot be ignored and further assessment is needed.

Assessment of the genotoxic potential of mintlactone.

Mintlactone (chemical name 3,6-dimethyl-5,6,7,7a-tetrahydro-1-benzofuran-2(4H)-one, CAS Number 13341-72-5) is a fragrance and flavor ingredient with reported uses in many different cosmetics, personal care, and household products. In order to evaluate the genotoxic potential of mintlactone, in vitro and in vivo genotoxicity tests were conducted. Results from bacterial mutagenicity tests varied across different batches of differing purity with positive results observed in TA98 only. An in vivo comet assay was also considered to be positive in livers of female mice but negative in male mice. In contrast, in vitro and in vivo micronucleus tests, as well as 3D skin comet/micronucleus tests, were negative, indicating no chromosomal or DNA damage. The underlying causes for these contradictory results are not clear. It appears that the purity and/or stability of the test material may be an issue. In the absence of dependable scientific information on the purity and/or storage stability of mintlactone, its safety for use as a fragrance ingredient cannot be substantiated.

A beta-glucosidase of an insect herbivore determines both toxicity and deterrence of a dandelion defense metabolite.

Gut enzymes can metabolize plant defense compounds and thereby affect the growth and fitness of insect herbivores. Whether these enzymes also influence feeding preference is largely unknown. We studied the metabolization of taraxinic acid ß-D-glucopyranosyl ester (TA-G), a sesquiterpene lactone of the common dandelion (Taraxacum officinale) that deters its major root herbivore, the common cockchafer larva (Melolontha melolontha). We have demonstrated that TA-G is rapidly deglucosylated and conjugated to glutathione in the insect gut. A broad-spectrum M. melolontha ß-glucosidase, Mm_bGlc17, is sufficient and necessary for TA-G deglucosylation. Using cross-species RNA interference, we have shown that Mm_bGlc17 reduces TA-G toxicity. Furthermore, Mm_bGlc17 is required for the preference of M. melolontha larvae for TA-G-deficient plants. Thus, herbivore metabolism modulates both the toxicity and deterrence of a plant defense compound. Our work illustrates the multifaceted roles of insect digestive enzymes as mediators of plant-herbivore interactions.

Plants produce certain substances to fend off attackers like plant-feeding insects. To stop these compounds from damaging their own cells, plants often attach sugar molecules to them. When an insect tries to eat the plant, the plant removes the stabilizing sugar, 'activating' the compounds and making them toxic or foul-tasting. Curiously, some insects remove the sugar themselves, but it is unclear what consequences this has, especially for insect behavior. Dandelions, Taraxacum officinale, make high concentrations of a sugar-containing defense compound in their roots called taraxinic acid ß-D-glucopyranosyl ester, or TA-G for short. TA-G deters the larvae of the Maybug ­ a pest also known as the common cockchafer or the doodlebug ­ from eating dandelion roots. When Maybug larvae do eat TA-G, it is found in their systems without its sugar. However, it is unclear whether it is the plant or the larva that removes the sugar. A second open question is how the sugar removal process affects the behavior of the Maybug larvae. Using chemical analysis and genetic manipulation, Huber et al. investigated what happens when Maybug larvae eat TA-G. This revealed that the acidity levels in the larvae's digestive system deactivate the proteins from the dandelion that would normally remove the sugar from TA-G. However, rather than leaving the compound intact, larvae remove the sugar from TA-G themselves. They do this using a digestive enzyme, known as a beta-glucosidase, that cuts through sugar. Removing the sugar from TA-G made the compound less toxic, allowing the larvae to grow bigger, but it also increased TA-G's deterrent effects, making the larvae less likely to eat the roots. Any organism that eats plants, including humans, must deal with chemicals like TA-G in their food. Once inside the body, enzymes can change these chemicals, altering their effects. This happens with many medicines, too. In the future, it might be possible to design compounds that activate only in certain species, or under certain conditions. Further studies in different systems may aid the development of new methods of pest control, or new drug treatments.

In Vitro Evaluation of the Individual and Combined Cytotoxic and Estrogenic Effects of Zearalenone, Its Reduced Metabolites, Alternariol, and Genistein.

Mycotoxins are toxic metabolites of filamentous fungi. Previous studies demonstrated the co-occurrence of Fusarium and Alternaria toxins, including zearalenone (ZEN), ZEN metabolites, and alternariol (AOH). These xenoestrogenic mycotoxins appear in soy-based meals and dietary supplements, resulting in the co-exposure to ZEN and AOH with the phytoestrogen genistein (GEN). In this study, the cytotoxic and estrogenic effects of ZEN, reduced ZEN metabolites, AOH, and GEN are examined to evaluate their individual and combined impacts. Our results demonstrate that reduced ZEN metabolites, AOH, and GEN can aggravate ZEN-induced toxicity; in addition, the compounds tested exerted mostly synergism or additive combined effects regarding cytotoxicity and/or estrogenicity. Therefore, these observations underline the importance and the considerable risk of mycotoxin co-exposure and the combined effects of mycoestrogens with phytoestrogens.

Role of an ABCB11930_1931del TC gene mutation in a temporal cluster of macrocyclic lactone-induced neurologic toxicosis in cats associated with products labeled for companion animal use.

OBJECTIVE: To determine whether ABCB11930_1931del TC predisposed cats to macrocyclic-lactone toxicosis and the frequency of the ABCB11930_1931del TC gene mutation in banked feline DNA samples. SAMPLE: DNA samples from 5 cats presented for neurologic clinical signs presumed to be caused by exposure to macrocyclic lactones and 1,006 banked feline DNA samples. PROCEDURES: The medical history pertaining to 5 cats was obtained from veterinarians who examined, treated, or performed necropsies on them. The DNA from these 5 cats and 1,006 banked feline samples were analyzed for the presence of the ABCB11930_1931del TC genotype. RESULTS: 4 of the 5 cats with neurologic signs presumed to be associated with macrocyclic-lactone exposure were homozygous for ABCB11930_1931del TC. The other cat had unilateral vestibular signs not typical of macrocyclic-lactone toxicosis. The distribution of genotypes from the banked feline DNA samples was as follows: 0 homozygous for ABCB11930_1931del TC, 47 heterozygous for ABCB11930_1931del TC, and 959 homozygous for the wild-type ABCB1 allele. Among the 47 cats with the mutant ABCB1 allele, only 3 were purebred (Ragdoll, Russian Blue, and Siamese). CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested a strong relationship between homozygosity for ABCB11930_1931del TC and neurologic toxicosis after topical application with eprinomectin-containing antiparasitic products labeled for use in cats. Although this genotype is likely rare in the general cat population, veterinarians should be aware of this genetic mutation in cats and its potential for enhancing susceptibility to adverse drug reactions.

Gamma-Decanolactone Alters the Expression of GluN2B, A1 Receptors, and COX-2 and Reduces DNA Damage in the PTZ-Induced Seizure Model After Subchronic Treatment in Mice.

Gamma-decanolactone (GD) has been shown to reduce epileptic behavior in different models, inflammatory decreasing, oxidative stress, and genotoxic parameters. This study assessed the GD effect on the pentylenetetrazole (PTZ) model after acute and subchronic treatment. We evaluated the expression of the inflammatory marker cyclooxygenase-2 (COX-2), GluN2B, a subunit of the NMDA glutamate receptor, adenosine A1 receptor, and GD genotoxicity and mutagenicity. Male and female mice were treated with GD (300 mg/kg) for 12 days. On the tenth day, they were tested in the Hot Plate test. On the thirteenth day, all animals received PTZ (90 mg/kg), and epileptic behavior PTZ-induced was observed for 30 min. Pregabalin (PGB) (30 mg/kg) was used as a positive control. Samples of the hippocampus and blood were collected for Western Blotting analyses and Comet Assay and bone marrow to the Micronucleus test. Only the acute treatment of GD reduced the seizure occurrence and increased the latency to the first stage 3 seizures. Males treated with GD for 12 days demonstrated a significant increase in the expression of the GluN2B receptor and a decrease in the COX-2 expression. Acute and subchronic treatment with GD and PGB reduced the DNA damage produced by PTZ in males and females. There is no increase in the micronucleus frequency in bone marrow after subchronic treatment. This study suggests that GD, after 12 days, could not reduce PTZ-induced seizures, but it has been shown to protect against DNA damage, reduce COX-2 and increase GluN2B expression.

Afidopyropen: Challenges and impact of a toxicokinetic study designed to identify a point of inflection from dose-proportionality.

Afidopyropen is an insecticide that acts as a transient receptor potential vanilloid subtype (TRPV) channel modulator in chordotonal organs of target insects and has been assessed for a wide range of toxicity endpoints including chronic toxicity and carcinogenicity in rats and mice. The current study evaluates the toxicokinetic properties of afidopyropen and its plasma metabolites in rats at dose levels where the pharmacokinetics (PK) are linear and nonlinear in an attempt to identify a point of inflection. Based on the results of this study and depending on the analysis method used, the kinetically derived maximum dose (KMD) is estimated to be between 2.5 and 12.5 mg/kg bw/d with linearity observed at doses below 2.5 mg/kg bw/d. A defined point of inflection could not be determined. These data demonstrate that consideration of PK is critical for improving the dose-selection in toxicity studies as well as to enhance human relevance of the interpretation of animal toxicity studies. The study also demonstrates the technical difficulty in obtaining a defined point of inflection from in vivo PK data.

Maduramicin induces cardiotoxicity via Rac1 signaling-independent methuosis in H9c2 cells.

Maduramicin frequently induces severe cardiotoxicity in target and nontarget animals in clinic. Apoptotic and non-apoptotic cell death mediate its cardiotoxicity; however, the underlying non-apoptotic cell death induced by maduramicin remains unclear. In current study, a recently described non-apoptotic cell death "methuosis" caused by maduramicin was defined in mammalian cells. Rat myocardial cell H9c2 was used as an in vitro model, showing excessively cytoplasmic vacuolization upon maduramicin (0.0625-5 µg/mL) exposure for 24 h. Maduramicin-induced reversible cytoplasmic vacuolization of H9c2 cells in a time- and concentration-dependent manner. The vacuoles induced by maduramicin were phase lucent with single membrane and were not derived from the swelling of organelles such as mitochondria, endoplasmic reticulum, lysosome, and Golgi apparatus. Furthermore, maduramicin-induced cytoplasmic vacuoles are generated from micropinocytosis, which was demonstrated by internalization of extracellular fluid-phase marker Dextran-Alexa Fluor 488 into H9c2 cells. Intriguingly, these cytoplasmic vacuoles acquired some characteristics of late endosomes and lysosomes rather than early endosomes and autophagosomes. Vacuolar H+ -ATPase inhibitor bafilomycin A1 efficiently prevented the generation of cytoplasmic vacuoles and decreased the cytotoxicity of H9c2 cells triggered by maduramicin. Mechanism studying indicated that maduramicin activated H-Ras-Rac1 signaling pathway at both mRNA and protein levels. However, the pharmacological inhibition and siRNA knockdown of Rac1 rescued maduramicin-induced cytotoxicity of H9c2 cells but did not alleviate cytoplasmic vacuolization. Based on these findings, maduramicin induces methuosis in H9c2 cells via Rac-1 signaling-independent seriously cytoplasmic vacuolization.

RIFM fragrance ingredient safety assessment, hexadeca-1,5-lactone, CAS Registry Number 7370-44-7.

The existing information supports the use of this material as described in this safety assessment. Hexadeca-1,5-lactone was evaluated for genotoxicity, repeated dose toxicity, reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization, and environmental safety. Data from the target material and read-across analog hydroxynonanoic acid, δ-lactone (CAS # 3301-94-8) show that hexadeca-1,5-lactone is not expected to be genotoxic. Data from analog δ-decalactone (CAS # 705-86-2) provide a calculated Margin of Exposure (MOE) > 100 for the repeated dose and reproductive toxicity endpoints. Data from analog δ-octalactone (CAS # 698-76-0) show that there are no safety concerns for skin sensitization under the current declared levels of use. The phototoxicity/photoallergenicity endpoints were evaluated based on ultraviolet (UV) spectra; hexadeca-1,5-lactone is not expected to be phototoxic/photoallergenic. The local respiratory toxicity endpoint was evaluated using the Threshold of Toxicological Concern (TTC) for a Cramer Class I material; exposure is below the TTC (1.4 mg/day). For the hazard assessment based on the screening data, hexadeca-1,5-lactone is not Persistent, Bioaccumulative, and Toxic (PBT) as per the International Fragrance Association (IFRA) Environmental Standards. Hexadeca-1,5-lactone could not be risk screened as there were no reported volumes of use for either North America or Europe in the 2015 IFRA Survey.