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The transient postnatal increase in circulating leptin levels, known as leptin surge, may increase later susceptibility to diet-induced obesity in rodents. However, the source of leptin during the surge needs to be better characterized, and the long-term effects of leptin are contradictory. Characterization of the interaction of leptin with the genetic background, sex, and other factors is required. Here, we focused on the impact of circulating leptin levels and several related variables, measured in 2- and 4-week-old (i) obesity-prone C57BL/6 (B6) and (ii) obesity-resistant A/J mice. In total, 264 mice of both sexes were used. Posttranscriptionally controlled leptin secretion from subcutaneous white adipose tissue, the largest adipose tissue depot in mice pups, was the primary determinant of plasma leptin levels. When the animals were randomly assigned standard chow or high-fat diet (HFD) between 12 - 24 weeks of age, the obesogenic effect of HFD-feeding was observed in B6 but not A/J mice. Only leptin levels at 2 weeks, i.e., close to the maximum in the postnatal leptin surge, correlated with both body weight (BW) trajectory throughout the life and adiposity of the 24-week-old mice. Leptin surge explained 13 and 7 % of the variance in BW and adiposity of B6 mice and 9 and 35 % of the variance in these parameters in A/J mice, with a minor role of sex. Our results prove the positive correlation between the leptin surge and adiposity in adulthood, reflecting the fundamental biological role of leptin. This role could be compromised in obese subjects.

The genotoxic and clastogenic/aneugeneic potentials of four α,β-unsaturated aldehydes, 2-phenyl-2-butenal, nona-2-trans-6-cis-dienal, 2-methyl-2-pentenal, and p-methoxy cinnamaldehyde, which are used as fragrance materials, were assessed using the Chicken Egg Genotoxicity Assay (CEGA) and the Hen's egg micronucleus (HET-MN) assay, respectively. Selection of materials was based on their chemical structures and the results of their previous assessment in the regulatory in vitro and/or in vivo genotoxicity test battery. Three tested materials, 2-phenyl-2-butenal, nona-2-trans-6-cis-dienal, and 2-methyl-2-pentenal, were negative in both, CEGA and HET-MN assays. These findings were congruent with the results of regulatory in vivo genotoxicity assays. In contrast, p-methoxy cinnamaldehyde, which was also negative in the in vivo genotoxicity assays, produced evidence of DNA damage, including DNA strand breaks and DNA adducts in CEGA. However, no increase in the micronucleus formation in blood was reported in the HET-MN study. Such variation in responses between the CEGA and HET-MN assay can be attributed to differences in the dosing protocols. Pretreatment with a glutathione precursor, N-acetyl cysteine, negated positive outcomes produced by p-methoxy cinnamaldehyde in CEGA, indicating that difference in response observed in the chicken egg and rodent models can be attributed to rapid glutathione depletion. Overall, our findings support the conclusion that CEGA and/or HET-MN can be considered as a potential alternative to animal testing as follow-up strategies for assessment of genotoxic potential of fragrance materials with evidence of genotoxicity in vitro., (© 2024 Environmental Mutagenesis and Genomics Society.)

Background: Sodium-glucose cotransporter 2 inhibitors (SGLT-2i) are glucose-lowering agents used for the treatment of type 2 diabetes mellitus, which also improve heart failure and decrease the risk of cardiovascular complications. Epicardial adipose tissue (EAT) dysfunction was suggested to contribute to the development of heart failure. We aimed to elucidate a possible role of changes in EAT metabolic and inflammatory profile in the beneficial cardioprotective effects of SGLT-2i in subjects with severe heart failure., Methods: 26 subjects with severe heart failure, with reduced ejection fraction, treated with SGLT-2i versus 26 subjects without treatment, matched for age (54.0 ± 2.1 vs. 55.3 ± 2.1 years, n.s.), body mass index (27.8 ± 0.9 vs. 28.8 ± 1.0 kg/m 2 , n.s.) and left ventricular ejection fraction (20.7 ± 0.5 vs. 23.2 ± 1.7%, n.s.), who were scheduled for heart transplantation or mechanical support implantation, were included in the study. A complex metabolomic and gene expression analysis of EAT obtained during surgery was performed., Results: SGLT-2i ameliorated inflammation, as evidenced by the improved gene expression profile of pro-inflammatory genes in adipose tissue and decreased infiltration of immune cells into EAT. Enrichment of ether lipids with oleic acid noted on metabolomic analysis suggests a reduced disposition to ferroptosis, potentially further contributing to decreased oxidative stress in EAT of SGLT-2i treated subjects., Conclusions: Our results show decreased inflammation in EAT of patients with severe heart failure treated by SGLT-2i, as compared to patients with heart failure without this therapy. Modulation of EAT inflammatory and metabolic status could represent a novel mechanism behind SGLT-2i-associated cardioprotective effects in patients with heart failure., (© 2024. The Author(s).)

For genotoxic carcinogens, covalent binding to DNA is a critical initiating event in tumorigenesis. The present research investigated dose-effect relationships of three genotoxic carcinogens representing different structural classes, 2-acetylaminofluorene (2-AAF), benzo[a]pyrene (B[a]P) and quinoline (QUI), to assess the existence of no-observed-effect-levels (NOELs) for the formation of DNA adducts. Carcinogens were administered into the air sac of fertilized turkey eggs over wide dose ranges in three daily injections on days 22 to 24 of incubation. DNA adducts were measured in the fetal turkey livers by the 32 P-nucleotide postlabeling (NPL) assay. B[a]P and QUI produced DNA adducts in a dosage-related manner and exhibited NOELs at 0.65 and 0.35 mg/kg bw/day, respectively. In contrast, 2-AAF formed DNA adducts at all tested dosages down to 0.005 mg/kg bw/day. Benchmark dose (BMD) analysis identified the potencies of 2-AAF and QUI to be similar, while B[a]P was the least potent compound. Overall, findings in fetal turkey livers demonstrated that exposure levels to genotoxic compounds that do not result in DNA adducts can exist but are not evident with all carcinogens of this type. The use of mechanistic dose-effect studies for genotoxic endpoints can provide critical information for prioritization of concerns for risk assessment., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Tetyana Kobets reports financial support was provided by Firmenich Inc. Benjamin Smith reports financial support was provided by Singapore National Research Foundation Whitespace grant., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Commonly consumed foods and beverages can contain chemicals with reported carcinogenic activity in rodent models. Moreover, exposures to some of these substances have been associated with increased cancer risks in humans. Food-borne carcinogens span a range of chemical classes and can arise from natural or anthropogenic sources, as well as form endogenously. Important considerations include the mechanism(s) of action (MoA), their relevance to human biology, and the level of exposure in diet. The MoAs of carcinogens have been classified as either DNA-reactive (genotoxic), involving covalent reaction with nuclear DNA, or epigenetic, involving molecular and cellular effects other than DNA reactivity. Carcinogens are generally present in food at low levels, resulting in low daily intakes, although there are some exceptions. Carcinogens of the DNA-reactive type produce effects at lower dosages than epigenetic carcinogens. Several food-related DNA-reactive carcinogens, including aflatoxins, aristolochic acid, benzene, benzo[a]pyrene and ethylene oxide, are recognized by the International Agency for Research on Cancer (IARC) as causes of human cancer. Of the epigenetic type, the only carcinogen considered to be associated with increased cancer in humans, although not from low-level food exposure, is dioxin (TCDD). Thus, DNA-reactive carcinogens in food represent a much greater risk than epigenetic carcinogens., Competing Interests: The authors report no competing interests to declare.

DNA damage is an established initiating event in the mutagenicity and carcinogenicity of genotoxic chemicals. Accordingly, assessment of this endpoint is critical for chemicals which are being developed for use in humans. To assess the ability of the Chicken Egg Genotoxicity Assay (CEGA) to detect genotoxic pharmaceuticals, a set of 23 compounds with different pharmacological and reported genotoxic effects was tested for the potential to produce nuclear DNA adducts and strand breaks in the embryo-fetal livers using the 32 P-nucleotide postlabeling (NPL) and comet assays, respectively. Due to high toxicity, two aneugens, colchicine and vinblastine, and an autophagy inhibitor, hydroxychloroquine, could not be evaluated. Out of the 20 remaining pharmaceuticals, 10 including estrogen modulators, diethylstilbestrol and tamoxifen, antineoplastics cyclophosphamide, etoposide, and mitomycin C, antifungal griseofulvin, local anesthetics lidocaine and prilocaine, and antihistamines diphenhydramine and doxylamine, yielded clear positive outcomes in at least one of the assays. The antihypertensive vasodilator hydralazine and antineoplastics streptozotocin and teniposide, produced only DNA strand breaks, which were not dose-dependent, and thus, the results with these 3 pharmaceuticals were considered equivocal. No DNA damage was detected for 7 compounds, including the purine antagonist 6-thioguanine, antipyretic analgesics acetaminophen and phenacetin, antibiotic ciprofloxacin, antilipidemic clofibrate, anti-inflammatory ibuprofen, and sedative phenobarbital. However, low solubility of these compounds limited dosages tested in CEGA. Overall, results in CEGA were largely in concordance with the outcomes in other systems in vitro and in vivo, indicating that CEGA provides reliable detection of DNA damaging activity of genotoxic compounds. Further evaluations with a broader set of compounds would support this conclusion.

Background: Vertebrate meiotic recombination events are concentrated in regions (hotspots) that display open chromatin marks, such as trimethylation of lysines 4 and 36 of histone 3 (H3K4me3 and H3K36me3). Mouse and human PRDM9 proteins catalyze H3K4me3 and H3K36me3 and determine hotspot positions, whereas other vertebrates lacking PRDM9 recombine in regions with chromatin already opened for another function, such as gene promoters. While these other vertebrate species lacking PRDM9 remain fertile, inactivation of the mouse Prdm9 gene, which shifts the hotspots to the functional regions (including promoters), typically causes gross fertility reduction; and the reasons for these species differences are not clear., Results: We introduced Prdm9 deletions into the Rattus norvegicus genome and generated the first rat genome-wide maps of recombination-initiating double-strand break hotspots. Rat strains carrying the same wild-type Prdm9 allele shared 88% hotspots but strains with different Prdm9 alleles only 3%. After Prdm9 deletion, rat hotspots relocated to functional regions, about 40% to positions corresponding to Prdm9-independent mouse hotspots, including promoters. Despite the hotspot relocation and decreased fertility, Prdm9-deficient rats of the SHR/OlaIpcv strain produced healthy offspring. The percentage of normal pachytene spermatocytes in SHR-Prdm9 mutants was almost double than in the PWD male mouse oligospermic sterile mutants. We previously found a correlation between the crossover rate and sperm presence in mouse Prdm9 mutants. The crossover rate of SHR is more similar to sperm-carrying mutant mice, but it did not fully explain the fertility of the SHR mutants. Besides mild meiotic arrests at rat tubular stages IV (mid-pachytene) and XIV (metaphase), we also detected postmeiotic apoptosis of round spermatids. We found delayed meiosis and age-dependent fertility in both sexes of the SHR mutants., Conclusions: We hypothesize that the relative increased fertility of rat versus mouse Prdm9 mutants could be ascribed to extended duration of meiotic prophase I. While rat PRDM9 shapes meiotic recombination landscapes, it is unnecessary for recombination. We suggest that PRDM9 has additional roles in spermatogenesis and speciation-spermatid development and reproductive age-that may help to explain male-specific hybrid sterility.

Long transgenes are often used in mammalian genetics, e.g., to rescue mutations in large genes. In the course of experiments addressing the genetic basis of hybrid sterility caused by meiotic defects in mice bearing different alleles of Prdm9, we discovered that introduction of copy-number variation (CNV) via two independent insertions of long transgenes containing incomplete Prdm9 decreased testicular weight and epididymal sperm count. Transgenic animals displayed increased occurrence of seminiferous tubules with apoptotic cells at 18 days postpartum (dpp) corresponding to late meiotic prophase I, but not at 21 dpp. We hypothesized that long transgene insertions could cause asynapsis, but the immunocytochemical data revealed that the adult transgenic testes carried a similar percentage of asynaptic pachytene spermatocytes as the controls. These transgenic spermatocytes displayed less crossovers but similar numbers of unrepaired meiotic breaks. Despite slightly increased frequency of metaphase I spermatocytes with univalent chromosome(s) and reduced numbers of metaphase II spermatocytes, cytological studies did not reveal increased apoptosis in tubules containing the metaphase spermatocytes, but found an increased percentage of tubules carrying apoptotic spermatids. Sperm counts of subfertile animals inversely correlated with the transcription levels of the Psmb1 gene encoded within these two transgenes. The effect of the transgenes was dependent on sex and genetic background. Our results imply that the fertility of transgenic hybrid animals is not compromised by the impaired meiotic synapsis of homologous chromosomes, but can be negatively influenced by the increased expression of the introduced genes.

DNA-damaging activities of twenty-four structurally diverse unsubstituted and substituted cyclic compounds were assessed in embryo-fetal chicken livers. Formation of DNA adducts and strand breaks were measured using the nucleotide 32 P-postlabelling (NPL) and comet assays, respectively. Unsubstituted monocyclic benzene, polycyclic fused ring compound naphthalene, covalently connected polycyclic ring compound biphenyl, and heterocyclic ring compound fluorene did not produce DNA damage. Amino-substituted monocyclic compounds, aniline and p-phenylenediamine, as well as polycyclic 1-naphthylamine were also negative. In contrast, carcinogenic monocyclic methyl-substituted anilines: o-toluidine, 2,6-xylidine, 3,4-dimethylaniline, 4-chloro-o-toluidine; 2 methoxy-substituted methylaniline: p-cresidine; 2,4 and 2,6 diamino- or dinitro- substituted toluenes all produced DNA damage. Genotoxic polycyclic amino-substituted 2-naphthylamine, 4-aminobiphenyl, benzidine, methyl-substituted 3,2'-dimethyl-4-aminobiphenyl and 4-dimethylaminoazobenzene as well as amino- and nitro- fluorenes substituted at the 1 or 2 positions also were positive in at least one of the assays. Overall, the DNA damaging activity of cyclic compounds in embryo-fetal chicken livers reflected the type and position of the substitution on the aromatic ring. Additionally, substituted polycyclic compounds exhibited higher DNA-damaging potency compared to monocyclic chemicals. These results are congruent with in vivo findings in other species, establishing chicken eggs as a reliable system for structure-activity assessment of members of groups of related chemicals., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Formation of DNA adducts by five alkenylbenzenes, safrole, methyl eugenol, eugenol, and asarone with either α- or β-conformation, was analyzed in fetal avian livers in two in ovo models. DNA reactivity of the carcinogens safrole and methyl eugenol was previously demonstrated in the turkey egg model, whereas non-genotoxic eugenol was negative. In the current study, alkenylbenzenes were also tested in the chicken egg model. Injections with alkenylbenzenes were administered to fertilized turkey or chicken eggs for three consecutive days. Three hours after the last injection, liver samples were evaluated for DNA adduct formation using the 32 P-nucleotide postlabeling assay. DNA samples from turkey livers were also analyzed for adducts using mass spectrometry. In both species, genotoxic alkenylbenzenes safrole, methyl eugenol, α- and β-asarone produced DNA adducts, the presence and nature of which, with exception of safrole, were confirmed by mass spectrometry, validating the sensitivity of the 32 P-postlabeling assay. Overall, the results of testing were congruent between fetal turkey and chicken livers, confirming that these organisms can be used interchangeably. Moreover, data obtained in both models is comparable to genotoxicity findings in other species, supporting the usefulness of avian models for the assessment of genotoxicity as a potential alternative to animal models., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A hallmark of meiosis is the rearrangement of parental alleles to ensure genetic diversity in the gametes. These chromosome rearrangements are mediated by the repair of programmed DNA double-strand breaks (DSBs) as genetic crossovers between parental homologs. In mice, humans, and many other mammals, meiotic DSBs occur primarily at hotspots, determined by sequence-specific binding of the PRDM9 protein. Without PRDM9, meiotic DSBs occur near gene promoters and other functional sites. Studies in a limited number of mouse strains showed that functional PRDM9 is required to complete meiosis, but despite its apparent importance, Prdm9 has been repeatedly lost across many animal lineages. Both the reason for mouse sterility in the absence of PRDM9 and the mechanism by which Prdm9 can be lost remain unclear. Here, we explore whether mice can tolerate the loss of Prdm9 By generating Prdm9 functional knockouts in an array of genetic backgrounds, we observe a wide range of fertility phenotypes and ultimately demonstrate that PRDM9 is not required for completion of male meiosis. Although DSBs still form at a common subset of functional sites in all mice lacking PRDM9, meiotic outcomes differ substantially. We speculate that DSBs at functional sites are difficult to repair as a crossover and that by increasing the efficiency of crossover formation at these sites, genetic modifiers of recombination rates can allow for meiotic progression. This model implies that species with a sufficiently high recombination rate may lose Prdm9 yet remain fertile., (© 2019 Mihola et al.; Published by Cold Spring Harbor Laboratory Press.)

Leishmaniasis is a serious health problem in many countries, and continues expanding to new geographic areas including Europe and USA. This disease, caused by parasites of Leishmania spp. and transmitted by phlebotomine sand flies, causes up to 1.3 million new cases each year and despite efforts toward its functional dissection and treatment it causes 20-50 thousands deaths annually. Dependence of susceptibility to leishmaniasis on sex and host's genes was observed in humans and in mouse models. Several laboratories defined in mice a number of Lmr ( Leishmania major response ) genetic loci that control functional and pathological components of the response to and outcome of L. major infection. However, the development of its most aggressive form, visceral leishmaniasis, which is lethal if untreated, is not yet understood. Visceral leishmaniasis is caused by infection and inflammation of internal organs. Therefore, we analyzed the genetics of parasite load, spread to internal organs, and ensuing visceral pathology. Using a new PCR-based method of quantification of parasites in tissues we describe a network-like set of interacting genetic loci that control parasite load in different organs. Quantification of Leishmania parasites in lymph nodes, spleen and liver from infected F 2 hybrids between BALB/c and recombinant congenic strains CcS-9 and CcS-16 allowed us to map two novel parasite load controlling Leishmania major response loci, Lmr24 and Lmr27 . We also detected parasite-controlling role of the previously described loci Lmr4, Lmr11, Lmr13, Lmr14, Lmr15 , and Lmr25 , and describe 8 genetic interactions between them. Lmr14, Lmr15, Lmr25 , and Lmr27 controlled parasite load in liver and lymph nodes. In addition, Leishmania burden in lymph nodes but not liver was influenced by Lmr4 and Lmr24 . In spleen, parasite load was controlled by Lmr11 and Lmr13 . We detected a strong effect of sex on some of these genes. We also mapped additional genes controlling splenomegaly and hepatomegaly. This resulted in a systematized insight into genetic control of spread and load of Leishmania parasites and visceral pathology in the mammalian organism.

In the deliberations over many years on the question of thresholds for the carcinogenicity of chemicals, the dominant paradigm has been the linear no-threshold (LNT) model, derived from concepts formulated in radiation mutagenicity. Based on the analogy with radiation, the key mechanistic assumption underlying the assessment of the dose-effect of chemical-induced carcinogenicity has been that any dose, no matter how low, can lead to induction of mutations, which will result in some risk of neoplasia. The LNT assumption, however, was never well founded and, its application to chemical carcinogens, does not allow for differences in their disposition or mechanisms of action. These mechanisms include DNA-reactivity and epigenetic effects, resulting from very different properties of carcinogens, leading to different dose effects. This review of the research on dose effects of chemical carcinogens administered by repeat dosing for long duration reveals that only some experiments involving what are now recognized as DNA-reactive carcinogens yielded dose effects for induction of tumors which were consistent with the absence of a threshold (for 6/14 chemicals). None of these studies, however, included low doses documented not to produce genetic or other cellular toxicities that underlie carcinogenicity. Otherwise, most dose-effect experiments, including all with epigenetic agents (7), revealed no-observed-effect-levels for tumors, indicative of subthreshold doses. Based on highly informative experimental data, including relevant mechanistic data, it is concluded that no-effect-levels exist for both carcinogen-induced precursor effects and neoplasia. Accordingly, we conclude that, at non-toxic dosages, thresholds exist for the induction of experimental cancer by all types of carcinogens., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Exposure to environmental chemicals has been shown to have an impact on the epigenome. One example is a known human carcinogen 1,3-butadiene which acts primarily by a genotoxic mechanism, but also disrupts the chromatin structure by altering patterns of cytosine DNA methylation and histone modifications. Sex-specific differences in 1,3-butadiene-induced genotoxicity and carcinogenicity are well established; however, it remains unknown whether 1,3-butadiene-associated epigenetic alterations are also sex dependent. Therefore, we tested the hypothesis that inhalational exposure to 1,3-butadiene will result in sex-specific epigenetic alterations. DNA damage and epigenetic effects of 1,3-butadiene were evaluated in liver, lung, and kidney tissues of male and female mice of two inbred strains (C57BL/6J and CAST/EiJ). Mice were exposed to 0 or 425 ppm of 1,3-butadiene by inhalation (6 h/day, 5 days/week) for 2 weeks. Strain- and tissue-specific differences in 1,3-butadiene-induced DNA adducts and crosslinks were detected in the liver, lung and kidney; however, significant sex-specific differences in DNA damage were observed in the lung of C57BL/6J mice only. In addition, we assessed expression of the DNA repair genes and observed a marked upregulation of Mgmt in the kidney in female C57BL/6J mice. Sex-specific epigenetic effects of 1,3-butadiene exposure were evident in alterations of cytosine DNA methylation and histone modifications in the liver and lung in both strains. Specifically, we observed a loss of cytosine DNA methylation in the liver and lung of male and female 1,3-butadiene-exposed C57BL/6J mice, whereas hypermethylation was found in the liver and lung in 1,3-butadiene-exposed female CAST/EiJ mice. Our findings suggest that strain- and sex-specific effects of 1,3-butadiene on the epigenome may contribute to the known differences in cancer susceptibility.

Chemicals with carcinogenic activity in either animals or humans produce increases in neoplasia through diverse mechanisms. One mechanism is reaction with nuclear DNA. Other mechanisms consist of epigenetic effects involving either modifications of regulatory macromolecules or perturbation of cellular regulatory processes. The basis for distinguishing between carcinogens that have either DNA reactivity or an epigenetic activity as their primary mechanism of action is detailed in this review. In addition, important applications of information on these mechanisms of action to carcinogenicity testing and human risk assessment are discussed.

The Chicken Egg Genotoxicity Assay (CEGA) demonstrated responsiveness to various DNA-reactive chemicals requiring metabolic activation, which implies broad bioactivation capability. To assess potential metabolic competence, expression profiles of metabolic genes in the embryo-chicken fetal liver were determined using microarray technology. Fertilized chicken eggs were injected under the CEGA protocol with vehicle (deionized water [DW]), the activation-dependent carcinogens, diethylnitrosamine (DEN), and N-nitrosodiethanolamine (NDELA) at doses producing no effect on survival. Previously in CEGA, DEN produced DNA damage, whereas NDELA did not. Expressions of 463 genes known to encode for phase I and II of endo- and xenobiotic metabolism were detected on the array. DW did not affect the expression of the selected genes, deregulating less than 1% of them. In contrast, DEN at 2 mg/egg and NDELA at 4 mg/egg produced significant transcriptomic alterations, up-regulating up to 41% and down-regulating over 31% of studied genes. Both nitrosamines modulated the majority of the genes in a similar manner, sharing 64 up-regulated and 93 down-regulated genes with respect to control group, indicating similarity in the regulation of their metabolism by avian liver. Differences in gene expression between DEN and NDELA were documented for several phase I CYP 450 genes that are responsible for nitrosamine biotransformation, as well as for phase II genes that regulate detoxication reactions. These findings could underlie the difference in genotoxicity of DEN and NDELA in CEGA. In conclusion, the analysis of gene expression profiles in embryo-chicken fetal liver dosed with dialkylnitrosamines demonstrated that avian species possess a complex array of inducible genes coding for biotransformation.

Genotoxicity of flavor and fragrance materials was assessed in Turkey Egg Genotoxicity Assay (TEGA) using 32 P-nucleotide postlabeling (NPL) and comet assays to detect hepatic DNA adducts and strand breaks. Twenty materials having results in GADD45a-Gluc 'BlueScreen HC' genotoxicity assay, and standard in vitro and in vivo tests, were selected to evaluate the accuracy of TEGA. Quinoline (QUI) and 2-acetylaminofluorene (AAF) served as positive comparators. Two materials, p-tert-butyldihydrocinnamaldehyde (BDHCA) and methyl eugenol (MEU) produced DNA adducts. BDHCA, p-t-butyl-α-methylhydrocinnamic aldehyde (BMHCA), trans-2-hexenal (HEX) and maltol (MAL) produced DNA strand breaks. Fifteen other materials were negative in both assays. Based on reports of oxidative DNA damage induction by MAL and 4-hydroxy-2.5-dimethyl-3(2H) furanone (HDMF), modified comet assays were conducted. Positive comet findings for MAL were not confirmed, and only equivocal evidence of oxidative damage was found. Accordingly, MAL was judged to have equivocal genotoxicity in TEGA. HDMF was positive in modified comet assay, indicating an ability to produce oxidative DNA damage. TEGA showed modest concordance with results in regulatory in vitro assays. Findings in TEGA, with few exceptions, were concordant with the results of in vivo genotoxicity and carcinogenicity testing. Thus, TEGA is an attractive alternative model for the assessment of genotoxic potential of chemicals in vivo., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Interferon-induced GTPases [guanylate-binding proteins (GBPs)] play an important role in inflammasome activation and mediate innate resistance to many intracellular pathogens, but little is known about their role in leishmaniasis. We therefore studied expression of Gbp2b / Gbp1 and Gbp5 mRNA in skin, inguinal lymph nodes, spleen, and liver after Leishmania major infection and in uninfected controls. We used two different groups of related mouse strains: BALB/c, STS, and CcS-5, CcS-16, and CcS-20 that carry different combinations of BALB/c and STS genomes, and strains O20, C57BL/10 (B10) and B10.O20, OcB-9, and OcB-43 carrying different combinations of O20 and B10 genomes. The strains were classified on the basis of size and number of infection-induced skin lesions as highly susceptible (BALB/c, CcS-16), susceptible (B10.O20), intermediate (CcS-20), and resistant (STS, O20, B10, OcB-9, OcB-43). Some uninfected strains differed in expression of Gbp2b / Gbp1 and Gbp5 , especially of Gbp2b / Gbp1 in skin. Uninfected BALB/c and STS did not differ in their expression, but in CcS-5, CcS-16, and CcS-20, which all carry BALB/c-derived Gbp gene-cluster, expression of Gbp2b / Gbp1 exceeds that of both parents. These data indicate trans -regulation of Gbp s. Infection resulted in approximately 10× upregulation of Gbp2b / Gbp1 and Gbp5 mRNAs in organs of both susceptible and resistant strains, which was most pronounced in skin. CcS-20 expressed higher level of Gbp2b / Gbp1 than both parental strains in skin, whereas CcS-16 expressed higher level of Gbp2b / Gbp1 than both parental strains in skin and liver. This indicates a trans -regulation present in infected mice CcS-16 and CcS-20. Immunostaining of skin of five strains revealed in resistant and intermediate strains STS, CcS-5, O20, and CcS-20 tight co-localization of Gbp2b/Gbp1 protein with most L. major parasites, whereas in the highly susceptible strain, BALB/c most parasites did not associate with Gbp2b/Gbp1. In conclusion, expression of Gbp2b / Gbp1 and Gbp5 was increased even in organs of clinically asymptomatic resistant mice. It suggests a hidden inflammation, which might contribute to control of persisting parasites. This is supported by the co-localization of Gbpb2/Gbp1 protein and L. major parasites in skin of resistant and intermediate but not highly susceptible mice.

Exposure to acrylonitrile induces formation of tumors at multiple sites in rats, with females being more sensitive. The present study assessed possible mechanisms of acrylonitrile tumorigenicity, covalent DNA binding, DNA breakage, and oxidative DNA damage, in two target tissues, the brain and Zymbal's glands, of sensitive female Fischer (F344) and Sprague-Dawley (SD) rats. One group received acrylonitrile in drinking water at 100 ppm for 28 days. Two other groups were administered either acrylonitrile in drinking water at 100 ppm or drinking water alone for 27 days, followed by a single oral gavage dose of 11 mg/kg bw 14 C-acrylonitrile on day 28. A positive control group received a single dose of 5 mg/kg bw of 7- 14 C-benzo[a]pyrene, on day 27 following the administration of drinking water for 26 days. Using liquid scintillation counting, no association of radiolabeled acrylonitrile with brain DNA was found. In accelerator mass spectrometry analysis, the association of 14 C of acrylonitrile with DNA in brains was detected and was similar in both strains, which may reflect acrylonitrile binding to protein as well as to DNA. Nucleotide 32 P-postlabeling assay analysis of brain samples from rats of both strains yielded no evidence of acrylonitrile DNA adducts. Negative conventional comet assay results indicate the absence of direct DNA strand breaks in the brain and Zymbal's gland in both strains of rats dosed with acrylonitrile. In both rat strains, positive results in an enhanced comet assay were found only in brain samples digested with formamidopyrimidine-DNA glycosylase but not with human 8-hydroxyguanine-DNA glycosylase, indicating possible oxidative DNA damage, other than 8-oxodG formation. In conclusion, definitive evidence of DNA binding of acrylonitrile in the brain and Zymbal's gland was not obtained under the test conditions. A role for oxidative stress in tumorigenesis in the brain but not Zymbal's gland may exist.

Leishmaniasis is an infectious disease caused by protozoan parasites of the genus Leishmania . There is no vaccine against human leishmaniasis and the treatment of the disease would benefit from a broader spectrum and a higher efficacy of leishmanicidal compounds. We analyzed the leishmanicidal activity and the mechanism of action of the calcium ionophore, calcimycin. L. major promastigotes were coincubated with calcimycin and the viability of the cells was assessed using resazurin assay. Calcimycin displayed dose-dependent effect with IC 50 = 0.16  μ M. Analysis of propidium iodide/LDS-751 stained promastigotes revealed that lower concentrations of calcimycin had cytostatic effect and higher concentrations had cytotoxic effect. To establish the mechanism of action of calcimycin, which is known to stimulate activity of mammalian constitutive nitric oxide synthase (NOS), we coincubated L. major promastigotes with calcimycin and selective NOS inhibitors ARL-17477 or L-NNA. Addition of these inhibitors substantially decreased the toxicity of calcimycin to Leishmania promastigotes. In doing so, we demonstrated for the first time that calcimycin has a direct leishmanicidal effect on L. major promastigotes. Also, we showed that Leishmania constitutive Ca 2+ /calmodulin-dependent nitric oxide synthase is involved in the parasite cell death. These data suggest activation of Leishmania nitric oxide synthase as a new therapeutic approach.

Background: Sex influences susceptibility to many infectious diseases, including some manifestations of leishmaniasis. The disease is caused by parasites that enter to the skin and can spread to the lymph nodes, spleen, liver, bone marrow, and sometimes lungs. Parasites induce host defenses including cell infiltration, leading to protective or ineffective inflammation. These responses are often influenced by host genotype and sex. We analyzed the role of sex in the impact of specific gene loci on eosinophil infiltration and its functional relevance., Methods: We studied the genetic control of infiltration of eosinophils into the inguinal lymph nodes after 8 weeks of Leishmania major infection using mouse strains BALB/c, STS, and recombinant congenic strains CcS-1,-3,-4,-5,-7,-9,-11,-12,-15,-16,-18, and -20, each of which contains a different random set of 12.5% genes from the parental "donor" strain STS and 87.5% genes from the "background" strain BALB/c. Numbers of eosinophils were counted in hematoxylin-eosin-stained sections of the inguinal lymph nodes under a light microscope. Parasite load was determined using PCR-ELISA., Results: The lymph nodes of resistant STS and susceptible BALB/c mice contained very low and intermediate numbers of eosinophils, respectively. Unexpectedly, eosinophil infiltration in strain CcS-9 exceeded that in BALB/c and STS and was higher in males than in females. We searched for genes controlling high eosinophil infiltration in CcS-9 mice by linkage analysis in F 2 hybrids between BALB/c and CcS-9 and detected four loci controlling eosinophil numbers. Lmr14 (chromosome 2) and Lmr25 (chromosome 5) operate independently from other genes (main effects). Lmr14 functions only in males, the effect of Lmr25 is sex independent. Lmr15 (chromosome 11) and Lmr26 (chromosome 9) operate in cooperation (non-additive interaction) with each other. This interaction was significant in males only, but sex-marker interaction was not significant. Eosinophil infiltration was positively correlated with parasite load in lymph nodes of F 2 hybrids in males, but not in females., Conclusions: We demonstrated a strong influence of sex on numbers of eosinophils in the lymph nodes after L. major infection and present the first identification of sex-dependent autosomal loci controlling eosinophilic infiltration. The positive correlation between eosinophil infiltration and parasite load in males suggests that this sex-dependent eosinophilic infiltration reflects ineffective inflammation.

The use of a food substance is Generally Recognized as Safe (GRAS) through scientific procedures or experience based on common use in food. The pivotal data used for GRAS determination must be of common knowledge and should include evidence for safety under the conditions of intended use of the substance. Such evidence includes data on the identity and specifications of the substance, its properties of absorption, distribution, metabolism and excretion, and depending on the level of concern, data on genotoxicity, acute and subchronic toxicity, reproductive and developmental toxicity and carcinogenicity. Several alternative procedures can be used as the replacement for standard scientific procedures in order to improve the GRAS process., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Certain alkenylbenzenes (AB), flavoring chemicals naturally occurring in spices and herbs, are established to be cytotoxic and hepatocarcinogenic in rodents. The purpose of the present study was to determine the DNA damaging potential of key representatives of this class using the Turkey Egg Genotoxicity Assay. Medium white turkey eggs with 22- to 24-day-old fetuses received three injections of nine AB with different carcinogenic potentials: safrole (1, 2 mg/egg), methyl eugenol (2, 4 mg/egg), estragole (20, 40 mg/egg), myristicin (25, 50 mg/egg), elemicin (20, 50 mg/egg), anethole (5, 10 mg/egg), methyl isoeugenol (40, 80 mg/egg), eugenol (1, 2.5 mg/egg), and isoeugenol (1, 4 mg/egg). Three hours after the last injection, fetal livers were harvested for measurement of DNA strand breaks, using the comet assay and DNA adducts formation, using the nucleotide(3) (2)P-postlabeling assay. Estragole, myristicin, and elemicin induced DNA stand breaks. These compounds as well as safrole, methyl eugenol and anethole, at the highest doses tested, induced DNA adduct formation. Methyl isoeugenol, eugenol, and isoeugenol did not induce genotoxicity. The genotoxic AB all had the structural features of either a double bond in the alkenyl side chain at the terminal 2',3'-position, favorable to formation of proximate carcinogenic 1'-hydroxymetabolite or terminal epoxide, or the absence of a free phenolic hydroxyl group crucial for formation of a nontoxic glucuronide conjugate. In contrast, methyl isoeugenol, eugenol and isoeugenol, which were nongenotoxic, possessed chemical features, unfavorable to activation., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Over-expression of transferrin receptor 1 (TFRC) is observed in hepatocellular carcinoma (HCC); however, there is a lack of conclusive information regarding the mechanisms of this dysregulation. In the present study, we demonstrated a significant increase in the levels of TFRC mRNA and protein in preneoplastic livers from relevant experimental models of human hepatocarcinogenesis and in human HCC cells. Additionally, using the TCGA database, we demonstrated an over-expression of TFRC in human HCC tissue samples and a markedly decreased level of microRNA-152 (miR-152) when compared to non-tumor liver tissue. The results indicated that the increase in levels of TFRC in human HCC cells and human HCC tissue samples may be attributed, in part, to a post-transcriptional mechanism mediated by a down-regulation of miR-152. This was evidenced by a strong inverse correlation between the level of TFRC and the expression of miR-152 in human HCC cells (r = -0.99, p = 4. 7 × 10-9), and was confirmed by in vitro experiments showing that transfection of human HCC cell lines with miR-152 effectively suppressed TFRC expression. This suggests that miR-152-specific targeting of TFRC may provide a selective anticancer therapeutic approach for the treatment of HCC.

Bisphenol A (BPA), an industrial chemical used in the manufacture of polycarbonate and epoxy resins, binds to the nuclear estrogen receptor with an affinity 4-5 orders of magnitude lower than that of estradiol. We reported previously that "high BPA" [100,000 and 300,000 µg/kg body weight (bw)/day], but not "low BPA" (2.5-2700 µg/kg bw/day), induced clear adverse effects in NCTR Sprague-Dawley rats gavaged daily from gestation day 6 through postnatal day (PND) 90. The "high BPA" effects partially overlapped those of ethinyl estradiol (EE2, 0.5 and 5.0 µg/kg bw/day). To evaluate further the potential of "low BPA" to induce biological effects, here we assessed the global genomic DNA methylation and gene expression in the prostate and female mammary glands, tissues identified previously as potential targets of BPA, and uterus, a sensitive estrogen-responsive tissue. Both doses of EE2 modulated gene expression, including of known estrogen-responsive genes, and PND 4 global gene expression data showed a partial overlap of the "high BPA" effects with those of EE2. The "low BPA" doses modulated the expression of several genes; however, the absence of a dose response reduces the likelihood that these changes were causally linked to the treatment. These results are consistent with the toxicity outcomes., (Published by Elsevier Ltd.)

Furan is a heterocyclic organic compound produced in the chemical manufacturing industry and also found in a broad range of food products, including infant formulas and baby foods. Previous reports have indicated that the adverse biological effects of furan, including its liver tumorigenicity, may be associated with epigenetic abnormalities. In the present study, we investigated the persistence of epigenetic alterations in rat liver. Male F344 rats were treated by gavage 5 days per week with 8 mg furan/kg body weight (bw)/day for 90 days. After the last treatment, rats were divided randomly into 4 groups; 1 group of rats was sacrificed 24 h after the last treatment, whereas other groups were maintained without further furan treatment for an additional 90, 180, or 360 days. Treatment with furan for 90 days resulted in alterations in histone lysine methylation and acetylation, induction of base-excision DNA repair genes, suggesting oxidative damage to DNA, and changes in the gene expression in the livers. A majority of these furan-induced molecular changes was transient and disappeared after the cessation of furan treatment. In contrast, histone H3 lysine 9 and H3 lysine 56 showed a sustained and time-depended decrease in acetylation, which was associated with formation of heterochromatin and altered gene expression. These results indicate that furan-induced adverse effects may be mechanistically related to sustained changes in histone lysine acetylation that compromise the ability of cells to maintain and control properly the expression of genetic information., (Published by Oxford University Press on behalf of the Society of Toxicology 2014. This work is written by US Government employees and is in the public domain in the US.)

1,3-Butadiene (BD), a widely used industrial chemical and a ubiquitous environmental pollutant, is a known human carcinogen. Although genotoxicity is an established mechanism of the tumorigenicity of BD, epigenetic effects have also been observed in livers of mice exposed to the chemical. To better characterize the diverse molecular mechanisms of BD tumorigenicity, we evaluated genotoxic and epigenotoxic effects of BD exposure in mouse tissues that are target (lung and liver) and non-target (kidney) for BD-induced tumors. We hypothesized that epigenetic alterations may explain, at least in part, the tissue-specific differences in BD tumorigenicity in mice. We evaluated the level of N-7-(2,3,4-trihydroxybut-1-yl)guanine adducts and 1,4-bis-(guan-7-yl)-2,3-butanediol crosslinks, DNA methylation, and histone modifications in male C57BL/6 mice exposed to filtered air or 425 ppm of BD by inhalation (6 h/day, 5 days/week) for 2 weeks. Although DNA damage was observed in all three tissues of BD-exposed mice, variation in epigenetic effects clearly existed between the kidneys, liver, and lungs. Epigenetic alterations indicative of genomic instability, including demethylation of repetitive DNA sequences and alterations in histone-lysine acetylation, were evident in the liver and lung tissues of BD-exposed mice. Changes in DNA methylation were insignificant in the kidneys of treated mice, whereas marks of condensed heterochromatin and transcriptional silencing (histone-lysine trimethylation) were increased. These modifications may represent a potential mechanistic explanation for the lack of tumorigenesis in the kidney. Our results indicate that differential tissue susceptibility to chemical-induced tumorigenesis may be attributed to tissue-specific epigenetic alterations., (Published by Oxford University Press on behalf of the Society of Toxicology 2014. This work is written by US Government employees and is in the public domain in the US.)

The presence of furan in common cooked foods along with evidence from experimental studies that lifetime exposure to furan causes liver tumors in rats and mice has caused concern to regulatory public health agencies worldwide; however, the mechanisms of the furan-induced hepatocarcinogenicity remain unclear. The goal of the present study was to investigate whether or not long-term exposure to furan causes epigenetic alterations in rat liver. Treating of male Fisher 344 rats by gavage 5 days per week with 0, 0.92, 2.0, or 4.4 mg furan/kg body weight (bw)/day resulted in dose- and time-dependent epigenetic changes consisting of alterations in DNA methylation and histone lysine methylation and acetylation, altered expression of chromatin modifying genes, and gene-specific methylation. Specifically, exposure to furan at doses 0.92, 2.0, or 4.4 mg furan/kg bw/day caused global DNA demethylation after 360 days of treatment. There was also a sustained decrease in the levels of histone H3 lysine 9 and H4 lysine 20 trimethylation after 180 and 360 days of furan exposure, and a marked reduction of histone H3 lysine 9 and H3 lysine 56 acetylation after 360 days at 4.4 mg/kg bw/day. These histone modification changes were accompanied by a reduced expression of Suv39h1, Prdm2, and Suv4-20h2 histone methyltransferases and Ep300 and Kat2a histone acetyltransferases. Additionally, furan at 2.0 and 4.4 mg/kg bw/day induced hypermethylation-dependent down-regulation of the Rassf1a gene in the livers after 180 and 360 days. These findings indicate possible involvement of dose- and time-dependent epigenetic modifications in the furan hepatotoxicity and carcinogenicity.

Background: L. tropica can cause both cutaneous and visceral leishmaniasis in humans. Although the L. tropica-induced cutaneous disease has been long known, its potential to visceralize in humans was recognized only recently. As nothing is known about the genetics of host responses to this infection and their clinical impact, we developed an informative animal model. We described previously that the recombinant congenic strain CcS-16 carrying 12.5% genes from the resistant parental strain STS/A and 87.5% genes from the susceptible strain BALB/c is more susceptible to L. tropica than BALB/c. We used these strains to map and functionally characterize the gene-loci regulating the immune responses and pathology., Methods: We analyzed genetics of response to L. tropica in infected F2 hybrids between BALB/c×CcS-16. CcS-16 strain carries STS-derived segments on nine chromosomes. We genotyped these segments in the F2 hybrid mice and tested their linkage with pathological changes and systemic immune responses., Principal Findings: We mapped 8 Ltr (Leishmania tropica response) loci. Four loci (Ltr2, Ltr3, Ltr6 and Ltr8) exhibit independent responses to L. tropica, while Ltr1, Ltr4, Ltr5 and Ltr7 were detected only in gene-gene interactions with other Ltr loci. Ltr3 exhibits the recently discovered phenomenon of transgenerational parental effect on parasite numbers in spleen. The most precise mapping (4.07 Mb) was achieved for Ltr1 (chr.2), which controls parasite numbers in lymph nodes. Five Ltr loci co-localize with loci controlling susceptibility to L. major, three are likely L. tropica specific. Individual Ltr loci affect different subsets of responses, exhibit organ specific effects and a separate control of parasite load and organ pathology., Conclusion: We present the first identification of genetic loci controlling susceptibility to L. tropica. The different combinations of alleles controlling various symptoms of the disease likely co-determine different manifestations of disease induced by the same pathogen in individual mice.

Children with asthma in remission had a significant increase in VC, FVC, FEV1, PEF, FEF25-FEF75 during prolonged sanatorium rehabilitation (for a year) in case of inclusion in the complex of rehabilitation respiration training "PARI PEP 2". Children who received the standard course of spa treatment don't have same results. Improvement intervals in PQ and QT(c) were demonstrated. It indicates a decrease in the risk of life-threatening arrhythmias. The simulator "PARI PEP 2" should be included in complex rehabilitation of children with asthma.

Nonalcoholic fatty liver disease (NAFLD) is a major health problem and a leading cause of chronic liver disease in the United States and developed countries. In humans, genetic factors greatly influence individual susceptibility to NAFLD. The goals of this study were to compare the magnitude of interindividual differences in the severity of liver injury induced by methyl-donor deficiency among individual inbred strains of mice and to investigate the underlying mechanisms associated with the variability. Feeding mice a choline- and folate-deficient diet for 12 wk caused liver injury similar to NAFLD. The magnitude of liver injury varied among the strains, with the order of sensitivity being A/J ≈ C57BL/6J ≈ C3H/HeJ < 129S1/SvImJ ≈ CAST/EiJ < PWK/PhJ < WSB/EiJ. The interstrain variability in severity of NAFLD liver damage was associated with dysregulation of genes involved in lipid metabolism, primarily with a down-regulation of the peroxisome proliferator receptor α (PPARα)-regulated lipid catabolic pathway genes. Markers of oxidative stress and oxidative stress-induced DNA damage were also elevated in the livers but were not correlated with severity of liver damage. These findings suggest that the PPARα-regulated metabolism network is one of the key mechanisms determining interstrain susceptibility and severity of NAFLD in mice.

Background: Leishmaniasis is a disease caused by protozoan parasites of genus Leishmania. The frequent involvement of Leishmania tropica in human leishmaniasis has been recognized only recently. Similarly as L. major, L. tropica causes cutaneous leishmaniasis in humans, but can also visceralize and cause systemic illness. The relationship between the host genotype and disease manifestations is poorly understood because there were no suitable animal models., Methods: We studied susceptibility to L. tropica, using BALB/c-c-STS/A (CcS/Dem) recombinant congenic (RC) strains, which differ greatly in susceptibility to L. major. Mice were infected with L. tropica and skin lesions, cytokine and chemokine levels in serum, and parasite numbers in organs were measured., Principal Findings: Females of BALB/c and several RC strains developed skin lesions. In some strains parasites visceralized and were detected in spleen and liver. Importantly, the strain distribution pattern of symptoms caused by L. tropica was different from that observed after L. major infection. Moreover, sex differently influenced infection with L. tropica and L. major. L. major-infected males exhibited either higher or similar skin pathology as females, whereas L. tropica-infected females were more susceptible than males. The majority of L. tropica-infected strains exhibited increased levels of chemokines CCL2, CCL3 and CCL5. CcS-16 females, which developed the largest lesions, exhibited a unique systemic chemokine reaction, characterized by additional transient early peaks of CCL3 and CCL5, which were not present in CcS-16 males nor in any other strain., Conclusion: Comparison of L. tropica and L. major infections indicates that the strain patterns of response are species-specific, with different sex effects and largely different host susceptibility genes.

Under laboratory conditions, hosts exposed twice to sand fly saliva are protected against severe leishmaniasis. However, people in endemic areas are exposed to the vector over a long term and may experience sand fly-free periods. Therefore, we exposed mice long- or short-term to Phlebotomus duboscqi bites, followed by Leishmania major infection either immediately or after a sand fly-free period. We showed that protection against leishmaniasis is limited to short-term exposure to sand flies immediately before infection. Our results may explain the persistence of leishmaniasis in endemic areas and should be taken into account when designing anti-Leishmania vaccines based on sand fly saliva., (Copyright © 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

This protocol describes an improved and optimized PCR-ELISA method for detection and quantification of Leishmania parasites in host tissues. Unlike other DNA-based assays, this method uses digoxigenin- and biotin-labeled primers. This eliminates the need for a separate step of hybridization of the PCR product with labeled probes. The PCR product is detected using sandwich ELISA with antidigoxigenin-detecting antibodies. Primers are complementary to the kinetoplast minicircle conserved region of parasite DNA, allowing the detection of several Leishmania species. For measurement of a wide range of parasite concentrations, +/-25 cycles were optimal. The sensitivity of this technique is 0.3 fg of parasite DNA per reaction in 40-cycle PCR-ELISA, corresponding to 0.004 parasites. DNA preparation by a standard TRI reagent procedure takes about 4 h. When DNA is prepared, a single person can test a large number of samples (at least 150) in a maximum of 7 h. This method might also be suitable for detecting and quantifying other pathogens, especially for detecting small differences in pathogen numbers.