Tetyana Kobets

Interstrain differences in the severity of liver injury induced by a choline- and folate-deficient diet in mice are associated with dysregulation of genes involved in lipid metabolism

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.—Tryndyak, V., de Conti, A., Kobets, T., Kutanzi, K., Koturbash, I., Han, T., Fuscoe, J. C., Latendresse, J. R., Melnyk, S., Shymonyak, S., Collins, L., Ross, S. A., Rusyn, I., Beland, F. A., Pogribny, I. P. Interstrain differences in the severity of liver injury induced by a choline‐ and folate‐deficient diet in mice are associated with dysregulation of genes involved in lipid metabolism. FASEB J. 26, 4592–4602 (2012). www.fasebj.org

Dose- and Time-Dependent Epigenetic Changes in the Livers of Fisher 344 Rats Exposed to Furan

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.

Persistence of furan-induced epigenetic aberrations in the livers of F344 rats

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.

Epigenetic Events Determine Tissue-Specific Toxicity of Inhalational Exposure to the Genotoxic Chemical 1,3-Butadiene in Male C57BL/6J Mice

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.

MicroRNA-152-mediated dysregulation of hepatic transferrin receptor 1 in liver carcinogenesis

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.

A no observed adverse effect level for DNA adduct formation in rat liver with prolonged dosing of the hepatocarcinogen 2-acetylaminofluorene

We have previously reported that the DNA-reactive hepatocarcinogen 2-acetylaminofluorene (AAF) produced in rat liver several key effects that were less than linear over a range of repeat doses, and that at low cumulative doses, no-adverse-effect-levels (NOAEL) were observed for several effects including hepatocellular cytotoxicity, enhanced cell proliferation, induction of preneoplastic foci and promotable liver neoplasms, although DNA adducts were still formed at dosages below the lowest NOAEL of 28 mg kg−1 cumulative dose for these other effects. This report details two further dose-effect studies at lower repeat doses than those previously used as well as identification of the specific types of DNA adducts formed. AAF was administered orally to male F344 rats by gavage at repeat dosages which in one experiment (A) ranged from 0.01 to 2.24 mg kg−1 bw per day, 7 days per week for 12 weeks followed by recovery for 4 weeks, and in a second (B) at lower dosages of 0.0026 or 0.026 mg kg−1 bw per day 3 days per week for 16 weeks. Initially the nonacetylated guanine adduct, N-(deoxyguanosine-8-yl)-aminofluorene, predominated. With continued dosing, the pattern of adducts changed such that by 4 weeks more acetylated, N-(deoxyguanosine-N2-yl)-AAF and N-(deoxyguanosine-8-yl)-AAF, adducts were present. In experiment A, total adducts reached a maximum by 12 weeks with levels of 6.0 adducts per 108 nucleotides at the lowest dosage. In experiment B, the total DNA adducts at the lowest dosage was below the limit of detection at 12 weeks, and at 0.6 in 108 nucleotides at 16 weeks, levels within the background range of 1.0–3.1 per 108 nucleotides. Thus, the cumulative dose of 0.125 mg kg−1 bw over 16 weeks was a NOAEL for adducts and hence would be predicted to be a threshold for hepatocarcinogenicity.

Chicken egg fetal liver DNA and histopathologic effects of structurally diverse carcinogens and non-carcinogens

Chicken egg fetal livers were evaluated for histopathological changes produced by four genotoxic hepatocarcinogens: 2-acetylaminofluorene (AAF), aflatoxin B1 (AFB1), benzo[a]pyrene (BaP), diethylnitrosamine (DEN); four structurally related non- or weakly- carcinogenic comparators: fluorene (FLU), aflatoxin B2 (AFB2), benzo[e]pyrene (BeP), N-nitrosodiethanolamine (NDELA); two epigenetic hepatocarcinogens: clofibric acid (CFA), phenobarbital (PB); and the non-carcinogen, D-mannitol (MAN). CFA, PB and MAN were also assessed for formation of DNA adducts using the 32P nucleotide postlabeling (NPL) assay and for DNA breaks using the comet assay. CFA was also assessed in enhanced comet assay for oxidative DNA damage induction. Eggs were dosed on days 9- 11 of incubation. For genotoxicity evaluation, livers were collected 3h after the last dose. Liver qualitative histopathology assessment was performed on days 12 and 18 of incubation. CFA was negative for DNA adducts but yielded clear evidence of DNA breaks due to oxidative stress. PB and MAN produced no DNA adducts or breaks. Liver to body weight ratios were not affected in most groups, but were decreased in DEN groups, and increased after PB dosing. Livers from control groups, FLU, AFB2, BeP, NDELA, CFA, and MAN groups, displayed a typical hepatocellular trabecular pattern at both time points. In contrast, the four genotoxic carcinogens induced time- and dose- related interference with fetal liver cell processes of proliferation, migration and differentiation, leading to hepatocellular and cholangiocellular pleomorphic dysplasia and re-(de-) differentiation with distortion of the trabecular pattern. In addition, dosing with the high dose of DEN produced gallbladder agenesis. PB induced hepatocellular hypertrophy, interference with migration, expressed as distortion of the trabecular pattern, and a moderate cholangiocellular dysplasia. In summary, histopathological analysis of chicken fetal livers revealed developmental anomalies, as well as genotoxicity-induced and, in the case of PB, adaptive morphological changes. Thus, the model provides histopathological outcomes of molecular effects.

Assessment of DNA Binding and Oxidative DNA Damage by Acrylonitrile in Two Rat Target Tissues of Carcinogenicity: Implications for the Mechanism of Action

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 Zymbals 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 14C-acrylonitrile on day 28. A positive control group received a single dose of 5 mg/kg bw of 7-14C-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 14C 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 32P-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 Zymbals 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 Zymbals gland was not obtained under the test conditions. A role for oxidative stress in tumorigenesis in the brain but not Zymbals gland may exist.

Expression of Genes Encoding for Xenobiotic Metabolism After Exposure to Dialkylnitrosamines in the Chicken Egg Genotoxicity Alternative Model

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.

GRAS determination scientific procedures and possible alternatives.

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.