Phenobarbital induces progressive patterns of GC-rich and gene-specific altered DNA methylation in the liver of tumor-prone B6C3F1 mice.

Altered DNA methylation contributes to tumorigenesis by affecting gene expression in a heritable fashion. Phenobarbital (PB) is a nongenotoxic rodent carcinogen which induces global hypomethylation and regions of hypermethylation in mouse liver. Liver tumor-sensitive (B6C3F1) and -resistant (C57BL/6) male mice were administered 0.05% (wt/wt) PB in drinking water for 2 or 4 weeks, and a 2-week recovery was included following each dosing period. DNA was isolated from liver (target) and kidney (nontarget) tissues. The methylation status of GC-rich regions of DNA was assessed via methylation-sensitive restriction digestion, arbitrarily primedpolymerase chain reaction, and capillary electrophoretic separation of products. PB-induced regions of altered methylation (RAMs) which carry forward from an early to a later time point are more likely to be mechanistically relevant as compared to those that do not. Twelve of 69 RAMs (17%) present in B6C3F1 liver at 2 weeks were also seen at 4 weeks, while only 1 of the 123 RAMs (< 1%) present in C57BL/6 liver was seen at 4 weeks. In the B6C3F1 mice, 57 unique (as compared to the C57BL/6) regions of altered hepatic methylation (RAMs), predominantly hypomethylation, were observed at 2 weeks, increasing to 86 at 4 weeks. Changes in methylation were largely reversible. Altered methylation in liver was highly dissimilar to that of kidney. Following 4 weeks PB, bisulfite sequencing revealed hypomethylation of Ha-ras in B6C3F1, but not C57BL/6, which correlated with increased gene expression. These data indicate that (1) progressive, nonrandom changes in methylation provide an epigenetic mechanism underlying the ability of PB to cause mouse liver tumorigenesis and (2) susceptibility to tumorigenesis is related inversely to the capacity to maintain normal patterns of methylation.     

Apoptosis in stages of mouse hepatocarcinogenesis: failure to counterbalance cell proliferation and to account for strain differences in tumor susceptibility.

C3H/He and B6C3F1 show much higher liver cancer susceptibility than C57BL/6J mice. We studied the hypothesis that this difference might result from failure of apoptosis. Hepatocarcinogenesis was induced by a single dose of N-nitrosodiethylamine (NDEA), followed by phenobarbital (PB) for up to 90 weeks. We observed (1) earlier appearance of putative preneoplastic foci (PPF), hepatocellular adenoma (HCA), and carcinoma (HCC) in C3H/He than in C57Bl/6J mice and (2) an increase of hepatocellular DNA synthesis in C3H/He and C57Bl/6J mice, compared to normal liver, via PPF and HCA to HCC. PB enhanced DNA synthesis and growth of PPF, in the C3H/He strain only, and of HCA and HCC of both strains. Apoptoses were rare in unaltered livers as well as in preneoplastic lesions, but tended to increase in HCA and HCC of both strains. PB lowered apoptotic activity in PPF of C3H/He mice, but enhanced it in HCA and HCC of C57Bl/6J mice at late stages. In conclusion, the strain difference in growth rates of PPF and tumors is largely determined by higher rates of cell proliferation in C3H/He mice, with and without promotion by PB. Moreover, in C57Bl/6J mice the promoting effect of PB was restricted to HCA and HCC and was not seen in PPF. Apoptosis was generally low and was not a major cause of the strain difference in tumor susceptibility. In contrast with rat liver, inhibition of apoptosis appears to be a minor determinant of tumor promotion in mice.     

Orphan nuclear receptor constitutive active/androstane receptor-mediated alterations in DNA methylation during phenobarbital promotion of liver tumorigenesis.

Altered DNA methylation is an epigenetic mechanism that plays a key role in the carcinogenesis process, and the nongenotoxic rodent hepatocarcinogen phenobarbital (PB) alters the methylation status of DNA in mouse liver. The constitutive active/androstane nuclear receptor (CAR) mediates half of the PB-induced hepatic gene expression changes and it is essential for liver tumor promotion in PB-treated mice. Here, a technique involving methylation-sensitive restriction digestion, arbitrarily primed PCR, and capillary electrophoresis was utilized to detect PB-induced regions of altered DNA methylation (RAMs) in CAR wildtype (WT) mice that are sensitive to promotion by PB and resistant CAR knockout (KO) mice. The CAR WT mice developed preneoplastic lesions after 23 weeks of PB treatment (precancerous) and liver tumors after 32 weeks, while the CAR KO mice did not develop tumors (Y. Yamamoto, et al., 2004, Cancer Res. 64, 7197-7200). Our goal was to discern those RAMs which are playing important roles in tumor formation by comparing the RAMs that form in sensitive and resistant groups of mice. Using this novel approach, 42 unique RAMs were identified in the precancerous as compared to the CAR KO, 23-week PB-treated tissue. Of these 42 RAMs, 14 carried forward to the tumor tissue, and additionally, 104 total unique RAMs were observed in the tumor tissue. These results indicate that there are unique RAMs occurring in the sensitive CAR WT mice and that a portion of these are seen in both the precancerous and tumor tissue. We hypothesize that these unique RAMs may be facilitating the tumorigenesis process, and these data support the view that DNA methylation plays a causative role in PB-induced tumorigenesis.     

No increase of apoptosis in regressing mouse liver after withdrawal of growth stimuli or food restriction.

In short-term in vivo experiments, liver growth and regression in mice with high (C3H/He), intermediate (B6C3F1) or low (C57BL/6J) susceptibility to hepatocarcinogenesis was compared. Liver growth was induced by dietary administration of phenobarbital (PB; 750 ppm) or nafenopin (NAF; 500 ppm). PB or NAF treatment for 7 days produced moderate increases of liver DNA (15% or 25-28%, respectively) along with pronounced hypertrophy. Liver growth was strongest in C3H/He mice. Cessation of PB or NAF treatment led to a rapid regression of liver hypertrophy. However, the enhanced hepatic DNA content persisted for at least 2 weeks in all mouse strains. Apoptosis was not increased at any time after cessation of treatment in all strains. Food restriction to 60% of the ad libitum intake did not amplify either regression of liver hyperplasia or the occurrence of apoptosis. No strain difference in the occurrence of apoptosis was detected. Mouse hepatocytes in liver regressing after mitogen withdrawal do not enter apoptosis as readily as rat hepatocytes.     

乙醇对嗜酒和非嗜酒小鼠体重和肝脏组织形态的影响

目的比较乙醇对嗜酒和非嗜酒小鼠躯体损害的易感性。方法以近交系C57BL/6J小鼠(嗜酒者)和远交系ICR小鼠(非嗜酒者)为实验动物,采用强制性饮酒程序:饮酒组小鼠饮用乙醇溶液8d,乙醇浓度从3%递增至20%。每天检测小鼠体重、摄食量、饮液量和乙醇摄入量。饮用乙醇期间取小鼠肝脏用组织化学方法观察肝脏组织病理学变化。结果乙醇对嗜酒小鼠体重增长的抑制作用明显强于非嗜酒小鼠。乙醇对两组小鼠的摄食量均有抑制作用,抑制程度无显著差异。乙醇抑制非嗜酒小鼠的饮液量,但对嗜酒小鼠无影响。实验期间两组小鼠的饮酒量相当,但嗜酒小鼠的肝脏损伤更为严重。结论嗜酒小鼠对乙醇毒性作用的耐受性弱于非嗜酒小鼠。     

Effects of microsomal enzyme inducers in vivo and inhibitors in vitro on the covalent binding of benzo[a]pyrene metabolites to DNA catalyzed by liver microsomes from genetically responsive and nonresponsive mice.

The in vitro DNA binding of benzo[a]pyrene metabolites generated by mouse liver microsomes can be resolved into at least nine distinct peaks by elution of a Sephadex LH20 column with a water-methanol gradient. These peaks, representing metabolite-nucleoside complexes, are named A (most polar) through I (least polar). 3-Methylcholanthrene, 2,3,7,8-tetrachlorodibenzo-p-dioxin, phenobarbital, Aroclor 1254, pregnenolone-16α-carbonitrile, or ethanol was administered in vivo to genetically “responsive” C57BL/6N or “nonresponsive” DBA/2N mice, in an attempt to understand and identify increases or decreases in reactive BP intermediates that bind to DNA. Rises or falls in these peaks are also noted when liver microsomes from control or 3-methylcholanthrene-treated C57BL/6N or DBA/2N mice were incubated in vitro with [³H]benzo[a]pyrene and microsomal enzyme inhibitors such as α-naphthoflavone, metyrapone or cyclohexene oxide. All of our interpretations concerning the binding of metabolites to DNA are consistent with non-K-region oxygenation of benzo[a]pyrene being mediated predominantly by cytochrome(s) P₁-450 and K-region oxygenation of benzo[a]pyrene being catalysed predominantly by form(s) of P-450 other than P₁-450. All of the biological perturbations are consistent with the following assignments. The major reactive intermediate of benzo[a]pyrene contributing to each peak is suggested to be: peaks A and C, an unknown dihydrodiol oxide; peaks B, D, F and I, quinones oxygenated further (or quinone-derived free radicals); peak E, both cis- and tans-7,8-diol-9,10-epoxides; peak F′, the 7.8-oxide; peak G, the 4.5-oxide; and peak H, an unknown phenol oxide. The DNA nucleosides are not identified in this study. Of the ten peaks listed here, it is of interest that the major metabolite(s) contributing to eight of the peaks (all except peaks F' and G) involve(s) more than a single mono-oxygenation by forms of cytochrome P-450. All peaks, with the exception of peak G, appear to be predominantly associated with benzo[a]pyrene metabolism mediated by P₁-450 and, therefore, controlled by the Ah locus. The use of these microsomal enzyme inducers or inhibitors—combined with the underlying genetic predisposition of the individual, tissue, or cell culture system under study—demonstrates that the balance between P-450 and epoxide hydrase, and the ratio of each form of P-450 to the other forms of P-450, can influence markedly the quantity and quality of reactive intermediates of benzo[a]pyrene that bind to DNA.     

Mutagenic activation of 2,4-diaminoanisole and 2-aminofluorene in vitro by liver and kidney fractions from aromatic hydrocarbon responsive and nonresponsive mice.

The mutagenicity of the two carcinogenic arylamines 2,4-diaminoanisole (2,4-DAA) and 2-aminofluorene (AF) was compared using liver and kidney fractions from two aromatic hydrocarbon (3-methylcholanthrene, MC) responsive and two nonresponsive mouse strains. MC pretreatment of mice caused an increase in 2,4-DAA mutagenicity with liver fractions from all four strains; however, much higher increases were seen in the two responsive than in the two nonresponsive strains. Kidney fractions had very low basal 2,4-DAA mutagenic activity. MC treatment led to 14–27-fold increase in 2,4-DAA mutagenicity in the responsive C57BL/6/BOM (B6) strain, but not in any of the other strains. AF mutagenicity was increased with liver fractions from all four mouse strains, but to the greatest extent in the B6 mice. AF showed high basal mutagenic activity with kidney fractions from all four strains, but MC treatment did not cause any increase in AF mutagenicity in any of the strains. Thus, there was a clear difference in the pattern of metabolic activation of the two arylamines 2,4-DAA and AF by liver and kidney fractions in mice, both with respect to constitutive activities and to the response to aromatic hydrocarbons.     

Effects of intermittent exposure to aflatoxin B1 on DNA and RNA adduct formation in rat liver: dose-response and temporal patterns.

We studied the effects of intermittent exposure to aflatoxin B1 (AFB1) on hepatic DNA and RNA adduct formation. Fisher-344 male rats were fed 0.01, 0.04, 0.4, or 1.6 ppm of AFB1 intermittently for 8, 12, 16, and 20 weeks, alternating with 4 weeks of dosing and 4 weeks of rest. Other groups of rats were fed 1.6 ppm of AFB1 continuously for 4, 8, 12, and 16 weeks. Control rats received AFB1-free NIH-31 meal diet. AFB1-DNA and -RNA adducts were measured by HPLC with fluorescence detection. The data are presented as total DNA or RNA adducts. The DNA and RNA adduct levels increased or decreased depending on the cycles of dosing and rest. Rats removed from treatment 1 month after 1 or 2 dosing cycles (8 and 16 weeks of intermittent exposure) showed approximately a twofold decrease in DNA adduct levels and a two- to elevenfold decrease in RNA adduct levels compared with rats euthanized immediately after the last dosing cycle (12 and 20 weeks of intermittent exposure). Our data indicate that DNA and RNA adducts increased linearly, from 0.01 ppm to 1.6 ppm of AFB1 after 12 and 20 weeks of intermittent treatment. A linear dose response was also apparent for DNA but not for RNA adducts after 8 and 16 weeks of treatment. As biomarkers of exposure, AFB1-RNA adducts were three to nine times more sensitive than AFB1-DNA adducts but showed greater variability. These results suggest that binding of AFB1 to hepatic DNA is a linear function of the dose, regardless of the way this is administered. The dose-response relationship for RNA adducts depends on the length of the no-dosing cycles and on the turnover rate of RNA.     

Effects of phenobarbital, 3-methylcholanthrene and beta-naphthoflavone pretreatment on mouse liver microsomal enzymes and on metabolite patterns of benzo[a]pyrene

C57B1/6 (B6), C3H, and DBA/2 (D2) strains of mice were pretreated with either a single dose of 3-methylcholanthrene (3-MC), β-naphthoflavone (β-NF) or α-naphthoflavone (α-NF), or with daily injections of phenobarbital (PB) for 4 days; control mice were injected with corn oil or saline only. Comparisons were made of the patterns of hepatic microsomal-protein bands on acrylamide gel electropherograms as well as the patterns of individual metabolites of benzo[a]pyrene (BP) extracted by ethyl acetate from incubation systems using these microsomes. Hepatic microsomes from control mice of all three strains produced similar protein bands and BP metabolite profiles, with B6 mice having slightly higher yields of the 9,10-diol and phenols of BP. α-NF had no apparent effect on the protein bands or the yields of BP metabolites. Sodium dodecylsulfate-polyacrylamide gel electrophoresis of hepatic microsomes revealed that PB pretreatment was followed by increases in the intensities of five protein bands with molecular weights between 43,000 and 68,000. The yields of BP metabolites were increased 1.5- to 3-fold for all three strains of mice after PB pretreatment. Pretreatment with 3-MC or β-NF had no apparent effect on the hepatic microsomal enzymes of D2 mice, either in terms of protein bands or metabolites of BP. For B6 and C3H mice, pretreatment with 3-MC or β-NF resulted in the appearance of a new protein band easily detectable in the range between molecular weights 53,000 and 58,000, as well as in increases of all BP metabolites. Pretreatment of B6 and C3H mice with 3-MC caused 10-fold increases in the microsomal yields of the 7,8-diol, and 5-fold increases in the yields of BP phenols. Pretreatment with β-NF caused a 7-fold increase of the 7,8-diol catalyzed by the hepatic microsomes of B6 mice and a 4-fold increase in the 7,8-diol in C3H mice; the yields of phenols were increased about 2.5-fold for both B6 and C3H mice. The results suggest that the induced microsomal proteins with molecular weights between 43,000 and 58,000 may contain induced forms of cytochromes(s) P-450 that activate different carbon atoms of the BP molecule and, therefore, lead to different metabolite profiles.     

Evidence that inhibited prostatic epithelial bud formation in 2,3,7,8-tetrachlorodibenzo-p-dioxin-exposed C57BL/6J fetal mice is not due to interruption of androgen signaling in the urogenital sinus.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) inhibits the androgen-dependent processes by which the urogenital sinus (UGS) of fetal mice forms prostatic epithelial buds. This inhibition is mediated by aryl hydrocarbon receptors in UGS mesenchyme and causes prostate lobes to develop abnormally. Experiments were conducted to test the hypothesis that TCDD inhibits prostatic budding in C57BL/6J mice by inhibiting androgen signaling. In utero TCDD exposure sufficient to inhibit budding (5 microg/kg maternal dose on gestation day [GD] 13) had no effect on testicular testosterone content on GD 16 or 18. Nor did it inhibit the conversion of testosterone to 5alpha-dihydrotestosterone (DHT) by the UGS. Both hydroxyflutamide (OH-flutamide; a competitive androgen receptor antagonist) and TCDD inhibited prostatic epithelial budding by UGSs cultured in vitro with DHT. To determine if TCDD inhibits responsiveness to androgens, primary mesenchymal cells prepared from UGSs cultured for three days with DHT were transiently transfected with an androgen-responsive reporter plasmid (MMTV-luciferase). OH-flutamide prevented DHT from increasing luciferase activity in these cells but TCDD did not. The same results were obtained when the mesenchymal cells were isolated from UGSs cultured with both DHT and TCDD. The lack of effect of TCDD on androgen-dependent gene expression was not due to inability of transfected UGS mesenchymal cells to respond to TCDD, as shown by significant increases in luciferase activity after transfection with plasmids containing CYP1A1 and CYP1B1 promoters. Finally, while OH-flutamide prevented DHT from altering androgen receptor and 5alpha-reductase type II mRNA expression in UGS organ culture, TCDD had no such effects. Collectively, these results suggest that TCDD inhibits prostatic epithelial bud formation without impairing the androgen receptor signaling pathway.     

Induction of liver microsomal cytochrome P-450 And associated monooxygenases by octachlorostyrene in inbred strains of mice: Lack of correlation with the murinE Ah locus

Single intraperitoneal injections of octachlorostyrene (OCS) and hexachlorobenzene in genetically polycyclic aromatic hydrocarbon ‘responsive’ C57/BL/6 (B6) mice led to a time- and dose-dependent increase in the levels of liver microsomal cytochromes P-450 and b₅ as well as in the activities of NADPH cytochrome P-450 (cytochrome c) reductase, ethylmorphine (EM) N-demethylase, 4-nitroanisole (PNA) O-demethylase and acetanilide 4-hydroxylase (AcA hydroxylase). No, or only a very moderate, increase in the activity of aryl hydrocarbon hydroxylase was seen after OCS and HCB, respectively. Pretreatments with phenobarbital (PB) or 3-methylcholanthrene (MC) both increased AcA hydroxylase activity to a similar degree, whereas pretreatment with polychlorinated biphenyls (Aroclor 1254) had an effect equal to the sum of PB and MC. Judged from sodium dodecylsulfate polyacrylamide gel electrophoresis studies, OCS and HCB predominantly increased a microsomal polypeptide of apparent mol. wt 52,000, similar to PB. A reduced response was seen after OCS or HCB treatment of aromatic hydrocarbon ‘non-responsive’ DBA/2 (D2) mice compared to B6 mice, both with respect to AcA hydroxylase as well as EM demethylase and PNA demethylase activities. OCS treatment of B6D2F1 mice resulted in a doubling of AcA hydroxylase activity, but in mice of the (B6D2)D2 backcross no distinct subgroupings of individual AcA hydroxylase activities were apparent. These results demonstrate that OCS is an inducer of the PB-type in mice and that induction of AcA hydroxylase by OCS is not regulated by the Ah locus.     

Mutagenesis of certain benzo(a)pyrene phenols in vitro following further metabolism by mouse liver.

Low concentrations of 1-hydroxy- and 3-hydroxybenzo[a]pyrene in the presence of NADPH and liver S-9 fraction from 3-methylcholanthrene-treated C57BL/6N mice are as much as 3-fold more mutagenic than benzo[a]pyrene in the bacteria Salmonella typhimurium LT₂ tester strain TA98. The level of mutagenicity rises with increasing phenol or S-9 protein concentration. In this system, 9-hydroxy-benzo[a]pyrene is slightly mutagenic, while 2-hydroxy-, 7-hydroxy- and 12-hydroxybenzo[a]pyrene are not mutagenic at low concentrations. The S-9 fraction from 3-methylcholanthrene-treated DBA/2N mice or phenobarbital-treated C 5 7BL/6N mice does not support significant levels of mutagenesis. The high level of mutagenicity by 1-hydroxy- or 3-hydroxybenzo[a]pyrene is inhibited by α-naphthoflavone but is not inhibited by metyrapone, 1, 2-epoxy-3, 3, 3-trichloropropane or glutathione. The substrate for UDP-glucuron-osyltransferase, UDP-glucuronic acid, prevents more than half of the mutagenicity caused by the further metabolism of 1-hydroxy- and 3-hydroxybenzot alpyrene. The combination of UDP-glucuronic acid and UDP-N-acetylglucosamine provides an even higher level of protection. The addition of the substrate for sulfotransferase(s), 3'-phosphoadenosine 5'-phosphate sulfate, also prevents about half of the mutagenesis caused by 1-hydroxy- or 3-hydroxybenzo[a]pyrene.     

Effects of beta-estradiol and bisphenol A on heat shock protein levels and localization in the mouse uterus are antagonized by the antiestrogen ICI 182,780.

Bisphenol A (BPA) exhibits many estrogen-like effects in the rodent uterus, but not all of these can be attenuated by antiestrogens. This suggests the involvement of alternate pathways of BPA action that do not involve the estrogen receptor (ER). An examination of the in vivo effects of BPA on uterine gene expression and protein levels should contribute to an understanding of its mechanism of action. In this study we examined the dose-related effects of BPA on levels of a suite of heat shock proteins (hsps) and on the localization of hsp90alpha, a chaperone of the ER, in uteri of ovariectomized B6C3F1 mice and compared these effects with those of beta-estradiol (E2). The antiestrogen ICI 182,780 (ICI) was co-administered with BPA or E2 in order to examine the potential role of the ER. BPA, although less potent than E2, increased hsp90alpha and grp94 to similar levels, but was much less effective than E2 in increasing levels of hsp72. Treatment with 100 mg BPA/kg/day or 2 microg E2/kg/day increased hsp90alpha to 300% of control levels and altered its tissue expression pattern. In uteri of corn oil (control)-treated mice, hsp90alpha predominantly localized in the cytoplasm and nuclei of epithelial cells. Upon treatment with BPA or E2 there was increased intensity of staining in the stroma and myometrium, and in the epithelium hsp90alpha was localized almost exclusively in the cytoplasm. The effects of BPA or E2 on hsp levels and hsp90alpha localization were attenuated by ICI. These results suggest an involvement of the ER in BPA- and E2-induced increases in uterine levels of hsp90alpha, grp94, and hsp72, and localization of hsp90alpha.     

Chemoprevention of liver cancer by plant polyphenols

Primary liver cancer or hepatocellular carcinoma (HCC) is one of the most frequent tumors representing the fifth commonest malignancy worldwide and the third cause of mortality from cancer. Currently, the treatments for HCC are not so effective and new strategies are needed for its fight. Chemoprevention, the use of natural or synthetic chemical agents to reverse, suppress or prevent carcinogenesis is considered an important way for confronting HCC. Many of the chemopreventive agents are phytochemicals, namely non-nutritive plant chemicals with protective or disease preventive properties. In this review, we focus on plant polyphenols, one of the most important classes of phytochemicals, their chemopreventive properties against HCC and discuss the molecular mechanisms accounting for this activity.