Levan Muskhelishvili

In Situ Hybridization and Immunohistochemical Analysis of Cytochrome P450 1B1 Expression in Human Normal Tissues

SUMMARY Cytochrome P450 1B1 (CYP1B1) is a recently cloned dioxin-inducible form of the cytochrome P450 supergene family of xenobiotic-metabolizing enzymes. CYP1B1 is constitutively expressed mainly in extrahepatic tissues and is inducible by aryl hydrocarbon receptor ligands. Human CYP1B1 is involved in activation of chemically diverse human procarcinogens, including polycyclic aromatic hydrocarbons and some aromatic amines, as well as the endogenous hormone 17β-estradiol. The metabolism of 17β-estradiol by CYP1B1 forms 4-hydroxyestradiol, a product believed to be important in estrogen-induced carcinogenesis. Although the distribution of CYP1B1 mRNA and protein in a number of human normal tissues has been well documented, neither the cells expressing CYP1B1 in individual tissue nor the intracellular localization of the enzyme has been thoroughly characterized. In this study, using nonradioactive in situ hybridization and immunohistochemistry, we examined the cellular localization of CYP1B1 mRNA and protein in a range of human normal tissues. CYP1B1 mRNA and protein were expressed in most samples of parenchymal and stromal tissue from brain, kidney, prostate, breast, cervix, uterus, ovary, and lymph nodes. In most tissues, CYP1B1 immunostaining was nuclear. However, in tubule cells of kidney and secretory cells of mammary gland, immunoreactivity for CYP1B1 protein was found in both nucleus and cytoplasm. This study demonstrates for the first time the nuclear localization of CYP1B1 protein. Moreover, the constitutive expression and wide distribution of CYP1B1 mRNA and protein in many human normal tissues suggest functional roles for CYP1B1 in the bioactivation of xenobiotic procarcinogens and endogenous substrates such as estrogens. (J Histochem Cytochem 49:229–236, 2001)

Methyl Deficiency, Alterations in Global Histone Modifications, and Carcinogenesis

The methyl-deficient model of endogenous hepatocarcinogenesis in rodents is unique in that dietary omission rather than the addition of chemical carcinogens leads to tumor formation. Thus, the biochemical and molecular events predisposing to cancer in this model result from chronic metabolic stress and provide an ideal model system to study progressive alterations that occur during carcinogenesis. Moreover, epigenetic alterations imposed by this diet are believed to be 1 of the main mechanisms responsible for malignant transformation of rat liver cells. In this study we examined the changes in global histone modification patterns in liver during hepatocarcinogenesis induced by methyl deficiency. Feeding animals the methyl-deficient diet (MDD) led to progressive loss of histone H4 lysine 20 trimethylation (H4K20me3), H3 lysine 9 trimethylation (H3K9me3), and histone H3 lysine 9 (H3K9ac) and histone H4 lysine 16 (H4K16ac) acetylation. A considerable decrease of H4K20me3 and H3K9ac was also detected in liver tumors induced by MDD. In contrast, liver tumors displayed an increase in H3K9me3 and H4K16ac. To determine the possible mechanism of alterations of histone modifications, we analyzed the expression of histone-modifying enzymes in liver during hepatocarcinogenesis. The expression of Suv4-20h2 and RIZ1 histone methyltransferases (HMTs) steadily decreased along with the development of liver tumors and reached its lowest level in tumor tissue, whereas the expression of Suv39-h1 HMT and histone acetyltransferase 1 (HAT1) substantially increased in tumors. These results illustrate the complexity and importance of histone modification changes in the etiology of hepatocarcinogenesis induced by MDD.

Increased distributional variance of mitochondrial DNA content associated with prostate cancer cells as compared with normal prostate cells.

Mitochondria are key organelles for apoptosis, and mitochondrial DNA (mtDNA) content can regulate cancer progression. Increases in mtDNA mutations and deletions have been reported in cancer; however, a detailed investigation of mtDNA content in cancer cells has not yet been conducted.

The tumor-promoting activity of 2-acetylaminofluorene is associated with disruption of the p53 signaling pathway and the balance between apoptosis and cell proliferation

The aromatic amine 2-acetylaminofluore (2-AAF) is a powerful complete genotoxic rat liver carcinogen that induces tumors without any additional interventions. While the tumor-initiating genotoxic activity of 2-AAF is well established, its tumor-promotion activity is far less understood. It is believed that the tumor-promoting property of 2-AAF is associated with selective enhancement of cell replication and sustained suppression of apoptosis in initiated cells. In the present study, we investigated the underlying mechanisms of tumor-promoting events induced by 2-AAF-exposure. Male Sprague-Dawley rats were fed NIH-31 diet containing 0.02% of 2-AAF for 12 and 24 weeks, and the expression pattern of genes associated with the p53-signaling pathway and microRNA genes was determined in the livers of control and 2-AAF-fed rats. The results indicate that the tumor-promoting property of 2-AAF during hepatocarcinogenesis is associated predominantly with the up-regulation of anti-apoptotic growth-related genes and down-regulation of expression of pro-apoptotic genes. This disrupts the balance between cell proliferation and apoptosis, which leads to consequential unrestricted cell proliferation, especially of initiated cells. Also, the long-term-administration of 2-AAF resulted in disruption of regulatory miR-34a-p53 feed-back loop that mediates apoptosis. This was evidenced by an increased expression of miR-34a in response to genotoxic effects of 2-AAF in the absence of p53 up-regulation, and loss of regulatory control of mir-34a on SIRT1 function. Additionally, the livers of 2-AAF-exposed rats were characterized by the substantial deregulation of expression of miR-18, miR-21, miR-182, and miR-200 family, microRNAs involved in control of apoptosis/cell proliferation and cell-cell contact pathways, two major pathways disrupted during the promotion stage of hepatocarcinogenesis.

Genetic and epigenetic changes in rat preneoplastic liver tissue induced by 2-acetylaminofluorene

Genotoxic carcinogens, including 2-acetylaminofluorene (2-AAF), in addition to exerting their genotoxic effects, often cause a variety of non-genotoxic alterations in cells. It is believed that these non-genotoxic effects may be indispensable events in tumorigenesis; however, there is insufficient knowledge to clarify the role of carcinogens in both the genetic and epigenetic changes in premalignant tissues and a lack of conclusive information on the link between epigenetic alterations and carcinogenic exposure. In the current study, we investigated whether or not the mechanism of 2-AAF-induced hepatocarcinogenesis consists of both genotoxic (genetic) and non-genotoxic (epigenetic) alterations. Male and female Sprague-Dawley rats were fed NIH-31 diet containing 0.02% of 2-AAF for 6, 12, 18 or 24 weeks. The levels of DNA adducts obtained from 2-AAF in liver and kidney tissues were assessed by high-performance liquid chromatography combined with electrospray tandem mass spectrometry (HPLC-ES-MS/MS). N-(Deoxyguanosine-8-yl)-2-aminofluorene was the major adduct detected at all time points in both tissues. Global DNA methylation in the livers and kidneys, as determined by an HpaII-based cytosine extension assay and by HPLC-ES-MS/MS, did not change over the 24-week period. In the livers of male rats, there was a progressive decrease of global and long interspersed nucleotide element-1-associated histone H4 lysine 20 trimethylation, as well as hypermethylation of the p16(INK4A) gene. These epigenetic changes were not observed in the livers of female rats or the kidneys of both sexes. Importantly, morphological evidence of formation and progression of neoplastic process was observed in the liver of male rats only. In conclusion, we have demonstrated that exposure of rats to genotoxic hepatocarcinogen 2-AAF, in addition to formation of 2-AAF-specific DNA lesions, resulted in substantial alterations in cellular epigenetic status.

Proliferating Cell Nuclear Antigen—A Marker for Ovarian Follicle Counts

Enumerating ovarian follicles is an effective way to estimate the extent of ovarian toxicity in female rodents exposed to xenobiotics. Differential follicle counts are useful in safety assessment bioassays and in interspecies extrapolation of ovarian toxicity. Counting the follicles in H&E-stained sections is labor intensive, tedious, and costly. In the present study we demonstrated that in rat formalin-fixed, paraffin-embedded ovary sections follicles of all degrees of maturity can be visualized by the use of antibody directed against proliferating cell nuclear antigen (PCNA). Follicles are easily distinguished from ovarian background with the ability to detect and identify primordial follicles being enhanced. This translates into a significant decrease in variability of follicle counts, labor, and cost. Specifically, variability dropped from 11% to 0.2%, the counting time was reduced by 46%, and the cost by 48%.

Epigenetic reprogramming of liver cells in tamoxifen-induced rat hepatocarcinogenesis.

Tamoxifen, a nonsteroidal anti‐estrogen, is a potent genotoxic hepatocarcinogen in rats, with both tumor initiating and promoting properties. Recently it has been demonstrated that genotoxic carcinogens, in addition to exerting genotoxic effects, often cause epigenetic alterations and these induced epigenetic changes may play important mechanistic role in carcinogenesis. In the present study, we investigated the role of tamoxifen‐induced epigenetic changes in hepatocarcinogenic process. The results of the study showed that exposure of female F344 rats to tamoxifen resulted in progressive loss of CpG methylation in regulatory sequences of long interspersed nucleotide elements (LINE‐1) and prominent increase in expression of LINE‐1 elements and c‐myc proto‐oncogene. The accumulation of tamoxifen‐induced DNA lesions was accompanied by the decreased level of Rad51, Ku70, and DNA polymerase β (Polβ) proteins that play a crucial role in maintenance of genomic stability. Furthermore, feeding rats with tamoxifen‐containing diet led to increased regenerative cell proliferation, as indicated by the increased level of Ki‐67 and proliferating cell nuclear antigen (PCNA) proteins. These data indicate that exposure of animals to genotoxic hepatocarcinogen tamoxifen led to early phenotypical alterations in livers characterized by emergence of epigenetically reprogrammed cells with a specific cancer‐related epigenetic phenotype prior to tumor formation.

The effects of subchronic acrylamide exposure on gene expression, neurochemistry, hormones, and histopathology in the hypothalamus–pituitary–thyroid axis of male Fischer 344 rats

Acrylamide (AA) is an important industrial chemical that is neurotoxic in rodents and humans and carcinogenic in rodents. The observation of cancer in endocrine-responsive tissues in Fischer 344 rats has prompted hypotheses of hormonal dysregulation, as opposed to DNA damage, as the mechanism for tumor induction by AA. The current investigation examines possible evidence for disruption of the hypothalamic-pituitary-thyroid axis from 14 days of repeated exposure of male Fischer 344 rats to doses of AA that range from one that is carcinogenic after lifetime exposure (2.5 mg/kg/d), an intermediate dose (10 mg/kg/d), and a high dose (50 mg/kg/d) that is neurotoxic for this exposure time. The endpoints selected include: serum levels of thyroid and pituitary hormones; target tissue expression of genes involved in hormone synthesis, release, and receptors; neurotransmitters in the CNS that affect hormone homeostasis; and histopathological evaluation of target tissues. These studies showed virtually no evidence for systematic alteration of the hypothalamic-pituitary-thyroid axis and do not support hormone dysregulation as a plausible mechanism for AA-induced thyroid cancer in the Fischer 344 rat. Specifically, there were no significant changes in: 1) mRNA levels in hypothalamus or pituitary for TRH, TSH, thyroid hormone receptor alpha and beta, as well 10 other hormones or releasing factors; 2) mRNA levels in thyroid for thyroglobulin, thyroid peroxidase, sodium iodide symporter, or type I deiodinases; 3) serum TSH or T3 levels (T4 was decreased at high dose only); 4) dopaminergic tone in the hypothalamus and pituitary or importantly 5) increased cell proliferation (Mki67 mRNA and Ki-67 protein levels were not increased) in thyroid or pituitary. These negative findings are consistent with a genotoxic mechanism of AA carcinogenicity based on metabolism to glycidamide and DNA adduct formation. Clarification of this mechanistic dichotomy may be useful in human cancer risk assessments for AA.

Coupling global methylation and gene expression profiles reveal key pathophysiological events in liver injury induced by a methyl-deficient diet

SCOPE A methyl-deficient diet induces liver injury similar to human nonalcoholic steatohepatitis, one of the main risk factors for the development of hepatocellular carcinoma. Previous studies have demonstrated that this diet perturbs DNA methylation by causing a profound loss of global cytosine methylation, predominantly at heavily methylated repetitive sequences. However, whether methyl deficiency affects the methylation status of gene promoters has not been explored. METHODS AND RESULTS Mouse gene expression and CpG island microarrays were used to characterize the gene expression and CpG island methylation profiles in the livers of C57BL/6J mice fed a methyl-deficient diet. We detected 164 genes that were differentially expressed and exhibited an inverse relationship between the gene expression and the extent of CpG island methylation. Furthermore, these genes were associated with altered lipid and glucose metabolism, DNA damage and repair, apoptosis, the development of fibrosis, and liver tissue remodeling. Although there were both increased and decreased levels of CpG island methylation, the number of hypomethylated genes was substantially greater than the number of hypermethylated genes. CONCLUSION The results this study demonstrate that pairing methylation profiles with gene expression profiles is a powerful approach to identify dysregulated high-priority fundamental pathophysiological pathways associated with disease development.

Interstrain differences in liver injury and one‐carbon metabolism in alcohol‐fed mice

Alcoholic liver injury is a major public health issue worldwide. Even though the major mechanisms of this disease have been established over the past decades, little is known about genetic susceptibility factors that may predispose individuals who abuse alcoholic beverages to liver damage and subsequent pathological conditions. We hypothesized that a panel of genetically diverse mouse strains may be used to examine the role of endoplasmic reticulum (ER) stress and one‐carbon metabolism in the mechanism of interindividual variability in alcoholic liver injury. We administered alcohol (up to 27 mg/kg/d) in a high‐fat diet using an intragastric intubation model for 28 days to male mice from 14 inbred strains (129S1/SvImJ, AKR/J, BALB/cJ, BALB/cByJ, BTBR T+tf/J, C3H/HeJ, C57BL/10J, DBA/2J, FVB/NJ, KK/HIJ, MOLF/EiJ, NZW/LacJ, PWD/PhJ, and WSB/EiJ). Profound interstrain differences (more than 3‐fold) in alcohol‐induced steatohepatitis were observed among the strains in spite of consistently high levels of urine alcohol that were monitored throughout the study. We found that ER stress genes were induced only in strains with the most liver injury. Liver glutathione and methyl donor levels were affected in all strains, albeit to a different degree. The most pronounced effects that were closely associated with the degree of liver injury were hyperhomocysteinemia and strain‐dependent differences in expression patterns of one‐carbon metabolism‐related genes. Conclusion: Our data demonstrate that strain differences in alcohol‐induced liver injury and steatosis are striking and independent of alcohol exposure and the most severely affected strains exhibit major differences in the expression of ER stress markers and genes of one‐carbon metabolism. (HEPATOLOGY 2012;56:130–139)