Evidence for cytochrome P-450 mediated metabolism in the bronchiolar damage by naphthalene

Intraperitoneal administration of the volatile hydrocarbon, naphthalene, resulted in severe bronchiolar epithelial cell necrosis in mice, while hepatic or renal necrosis was not observed. Pulmonary damage and mortality by naphthalene were increased by prior treatment with diethyl maleate and decreased by prior treatment with piperonyl butoxide (1600 mg/kg). SKF 525A pretreatment had no effect on naphthalene-induced pulmonary damage. Administration of [¹⁴C]naphthalene resulted in the covalent binding of radiolabel to tissue macromolecules. Highest levels of binding occurred in lung, liver and kidney. Levels of covalent binding reached a maximum 2–4 h after treatment and corresponded to rapid glutathione depletion in lung and liver. Covalent binding was dose-dependent and showed a threshold between 200 and 400 mg/kg which coincided with almost total depletion of tissue glutathione levels. Covalent binding of reactive metabolites was increased 3–4-fold by prior treatment with diethyl maleate, and was decreased 3–4-fold by pretreatment with piperonyl butoxide. These studies support the view that naphthalene-induced pulmonary damage is mediated by the cytochrome P-450-dependent metabolism of naphthalene and that glutathione plays an important role in the detoxification of the lung damaging metabolite(s).     

Proliferative response and renewal of hepatic function following cocaine administration in mice

Experiments were undertaken to investigate the hepatic, temporal and spatial sequence of events following a single injection of cocaine, a known hepatotoxin. Centrilobular necrosis was induced in male mice (DBA/2Ha) 24 hr post-injection (PI). The time course of hepatic damage was monitored by assaying microsomal cytochrome P450 content, the activity of microsomal FAD-containing monooxygenase (FAD-M) and by determining the levels of serum glutamic pyruvic transaminase (SGPT). Kinetics of the onset of DNA synthesis were determined by autoradiography of thin liver sections and the incorporation of 3H-methyl thymidine into perchloric-acid-precipitable material. There was no increase in the labelling index (LI) and thymidine (TdR) incorporation in the first 24 hr PI. The LI rose to 14.6% and TdR incorporation showed a 5-fold increase over control values 48 hr PI. Both indices declined slightly at 72 hr PI and returned to control values by 96 hr PI. In contrast, the cytochrome P450 content declined by 69%, the FAD-M activity dropped by 40% and the SGPT levels showed an 18-fold increase at 24 hr PI, coincident with cytological signs of necrosis. Although the patterns of recovery differed between these selected enzymes, normal values were attained by 96 hr PI. These results demonstrate that cell damage and hepatic dysfunction precede the onset of DNA synthesis and subsequent proliferation.     

Repair of DNA damage induced by anthanthrene, a polycyclic aromatic hydrocarbon (PAH) without bay or fjord regions

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants, formed during incomplete burning of coal, oil and gas. Several PAHs have carcinogenic and mutagenic potencies, but these compounds must be activated in order to exert their mutagenic effects. One of the principal pathways proposed for metabolic activation of PAHs involves the cytochrome P450 enzymes. The DNA damaging potential of cytochrome P450-activated PAHs is generally associated with their bay and fjord regions, and the DNA repair response of PAHs containing such regions has been thoroughly studied. However, little is known about the repair of DNA damage resulting from metabolites from PAHs without bay and fjord regions. We have investigated the six-ringed PAH anthanthrene (dibenzo[def,mno]chrysene), which does not posses bay or fjord motifs. We analyzed the repair profile of human cell extracts and of cell cultures in response to DNA damage induced by cytochrome P450-activated anthanthrene. In cell extracts, functional nucleotide excision repair (NER) and mismatch repair (MMR) activities were necessary to trigger a response to anthanthrene metabolite-induced DNA damage. In cell cultures, NER was responsible for the repair of anthanthrene metabolite-induced DNA damage. However, when the NER pathway was inactivated, a residual repair pathway performed the DNA repair.     

Evidence for oxidative metabolism in the genotoxicity of the atmospheric reaction product 2-nitronaphthalene in human lymphoblastoid cell lines.

2-Nitronaphthalene (2NN) has been identified as a mutagenic atmospheric reaction product of naphthalene in the Ames bacterial reversion assay. Recent experiments have shown this nitroarene to be genotoxic in a human lymphoblastoid cell line (MCL-5) transfected with plasmids encoding epoxide hydrolase and four cytochrome P450 monooxygenase activities. The present study investigated the genotoxicity of 2NN in two related human B-lymphoblastoid cell lines, h1A1v2 containing a single P450 isozyme (cytochrome P450 1A1) and L3 cells which are isogenic with MCL-5 cells and are distinguished only by the absence of transfected plasmids. The results indicate that 2NN-induced mutagenesis at the heterozygous thymidine kinase (tk) locus was dependent on metabolic activities provided by the transfected plasmids in MCL-5; no significant induction of mutants was observed in L3 cells studied in parallel. A similar induction of mutation was observed in h1A1v2 and MCL-5 cell lines at the tk locus and no induction was observed at the hemizygous hypoxanthine phosphoribosyl transferase (hprt) locus. The induction of mutations in h1A1v2 cells suggests that cytochrome P450 1A1 alone can activate 2NN to a mutagenic species, however, this interpretation may be confounded by differences between the h1A1v2 and MCL-5 cell lines. The observed genotoxic activity induced by 2NN prompted testing of the amino analogue, beta-naphthylamine (betaNA), to investigate potential similarities in the metabolic activation pathways of the two compounds. The negative response of betaNA in all cell lines suggests that 2NN and betaNA are not activated in these human cells by similar metabolic pathways.     

Steroid and xenobiotic effects on the adrenal cortex: mediation by oxidative and other mechanisms

Because steroids reach high concentrations within the adrenal cortex, effects of the direct interaction of steroids and cytochrome P450 enzymes are possible and may involve oxidative damage. Steroid pseudosubstrate effects studied in cultured adrenocortical cells show that these effects are probably not mediated by steroid receptors. Release of oxidants during pseudosubstrate interaction with cytochrome P450s may be responsible for loss of enzymatic activity observed; enzyme activity can be protected by cytochrome P450 inhibitors, antioxidants, and lowered oxygen concentration. There may be pathological effects of pseudosubstrates in the adrenal cortex. Cytochrome P450/pseudosubstrate effects could be involved in the aging and death of adrenocortical cells in vivo, and necrosis of the adrenal cortex due to excessive ACTH stimulation or due to the action of adrenolytic chemicals could result from damage by oxygen radicals originating from cytochrome P450s. The possible mechanism of damage to the adrenal cortex by the xenobiotics dimethylbenzanthracene, TCDD, 3-methylcholanthrene, and o', p'-DDD are reviewed.     

Induction and regulation of cytochrome P450 K-5 (lauric acid hydroxylase) in rat renal microsomes by starvation

The effects of starvation on rat renal cytochrome P-450s were studied. The content of spectrally measured cytochrome P-450 in the renal microsomes of male rats increased 2-fold with 72 h starvation, but cytochrome b5 and NADPH-cytochrome P-450 reductase were not induced. 7-Ethoxycoumarin O-dealkylation and aniline hydroxylation activities of the renal microsomes of control male rats were very low but were induced 2.5-3-fold by 72 h starvation. Aminopyrine N-demethylation and lauric acid hydroxylation activities were induced 1.5-2-fold by 72 h starvation. The changes in catalytic activities suggested that the contents of individual cytochrome P-450s in the renal microsomes were altered by starvation. The contents of some cytochrome P-450s were measured by Western blotting. P450 DM (P450IIE1), a typical form of cytochrome P-450 induced by starvation in rat liver, was barely detected in rat kidney and was induced 2-fold by 72 h starvation. P450 K-5, a typical renal cytochrome P-450 and lauric acid hydroxylase, accounted for 81% of the spectrally measured cytochrome P-450 in the renal microsomes of control male rats and was induced 2-fold by 72 h starvation. P450 K-5 was not induced in rat kidney by treatment with chemicals such as acetone or clofibrate. The renal microsomes of male rats contained 6-times as much P450 K-5 as those of female rats. These results suggest that P450 K-5 is regulated by an endocrine factor.     

Mouse pulmonary cytochrome P-450 naphthalene hydroxylase: cDNA cloning, sequence, and expression in Saccharomyces cerevisiae.

We have isolated a cDNA clone, Nah-2, encoding the cytochrome P-450Nah (naphthalene hydroxylase) from a mouse lung lambda ZAP cDNA library using anti-cytochrome P-450Nah IgG as a probe. This same antibody selectively blocked [Nagata, K., Martin, B.M., Gillette, J.R., & Sasame, H.A. (1990) Drug Metab. Dispos. 18, 557-564] the cytochrome P-450 in mouse lung microsomes that catalyzed the conversion of naphthalene to (1R,2S)-naphthalene 1,2-oxide, which has been postulated as a causative agent in the naphthalene-induced tissue-specific necrosis of Clara cells in mouse lung. The toxic effect is seen in mouse and not in rat. The cDNA encodes a polypeptide of 491 amino acids with a molecular mass of 50 kDa. Northern blot analysis with an Nah-2-specific probe revealed that the mRNA is expressed in a species- and tissue-specific manner, present only in mouse lung and liver and not in that of rat. The mRNA encoding Nah-2 is constitutively expressed and is not induced by either phenobarbital, pyrazole, pregnenolone 16 alpha-carbonitrile, or 3-methylcholanthrene. Comparative amino acid sequence analyses with other documented members of the P-450 gene superfamily revealed that this encoded protein is in the IIF subfamily. To analyze its substrate specificity, the cDNA was inserted into the vector, pAAH5, and expressed in the Saccharomyces cerevisiae strain, AH22. The presence of cytochrome P-450Nah in the microsomes isolated from transformed cells and analyzed by Western blot was confirmed by immunocomplexing product with anti-cytochrome P450Nah IgG. Furthermore, activity toward naphthalene in the microsomes from the transformed cells established that this clone encodes a naphthalene hydroxylase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of prior starvation on the development of acute thioacetamide-induced liver damage in rats

The effect of previous fasting on the liver morphological changes and microsomal cytochrome P-450 and b5 content was studied in thioacetamide-induced (100 mg/kg) rat liver necrosis. Starvation for 48 hours immediately before thioacetamide administration aggravates the dystrophic and necrotic processes, as revealed by histology, electron microscopic investigations and serum aminotransferase activity. The liver microsomal cytochrome P-450 concentration tended to decrease after thioacetamide challenge, with fasting resulting in a more significant loss of cytochrome P-450. Cytochrome b5 content, however, was found to increase in acute liver necrosis induced by thioacetamide.     

Proliferative Response and Renewal of Hepatic Function Following Cocaine Administration In Mice

ABSTRACT Experiments were undertaken to investigate the hepatic, temporal and spatial sequence of events following a single injection of cocaine, a known hepatotoxin. Centrilobular necrosis was induced in male mice (DBA/2Ha) 24 hr post-injection (PI). the time course of hepatic damage was monitored by assaying microsomal cytochrome P₄₅₀ content, the activity of microsomal FAD-containing monooxygenase (FAD-M) and by determining the levels of serum glutamic pyruvic transaminase (SGPT). Kinetics of the onset of DNA synthesis were determined by autoradiography of thin liver sections and the incorporation of ³H-methyl thymidine into perchloric-acid-precipitable material. There was no increase in the labelling index (LI) and thymidine (TdR) incorporation in the first 24 hr PI. the LI rose to 14.6% and TdR incorporation showed a 5-fold increase over control values 48 hr PI. Both indices declined slightly at 72 hr PI and returned to control values by 96 hr PI. In contrast, the cytochrome P₄₅₀ content declined by 69%, the FAD-M activity dropped by 40% and the SGPT levels showed an 18-fold increase at 24 hr PI, coincident with cytological signs of necrosis. Although the patterns of recovery differed between these selected enzymes, normal values were attained by 96 hr PI. These results demonstrate that cell damage and hepatic dysfunction precede the onset of DNA synthesis and subsequent proliferation.     

Prevention of naphthalene-induced pulmonary toxicity by glutathione prodrugs: roles for glutathione depletion in adduct formation and cell injury

Naphthalene is metabolized in the lung and liver to reactive intermediates by cytochrome P450 enzymes. These reactive species deplete glutathione, covalently bind to proteins, and cause necrosis in Clara cells of the lung. The importance of glutathione loss in naphthalene toxicity was investigated by using the glutathione prodrugs (glutathione monoethylester or cysteine-glutathione mixed disulfide) to maintain glutathione pools during naphthalene exposure. Mice given a single intraperitoneal injection of naphthalene (1.5 mmol/kg) were treated with either prodrug (2.5 mmol/kg) 30 min later. Both compounds effectively maintained glutathione levels and decreased naphthalene-protein adducts in the lung and liver. However, cysteine-glutathione mixed disulfide was more effective at preventing Clara cell injury. To study the prodrugs in Clara cells without the influence of hepatic naphthalene metabolism and circulating glutathione, dose-response and time-course studies were conducted with intrapulmonary airway explant cultures. Only the ester of glutathione raised GSH in vitro; however, both compounds limited protein adducts and cell necrosis. In vitro protection was not associated with decreased naphthalene metabolism. We conclude that (1) glutathione prodrugs can prevent naphthalene toxicity in Clara cells, (2) the prodrugs effectively prevent glutathione loss in vivo, and (3) cysteine-glutathione mixed disulfide prevents naphthalene injury in vitro without raising glutathione levels.     

Optimization of an in vivo plant P450 monooxygenase system in Saccharomyces cerevisiae

Cytochrome P450s are heme-thiolate oxygenases involved in a wide number of reactions such as epoxidation, hydroxylation, and demethylation. Heterologously expressed eukaryotic P450s are potentially useful biocatalysts for stereospecific oxygenation reactions under mild conditions. Numerous factors, such as intracellular pH, cytochrome P450, cytochrome P450 reductase, NADPH, and oxygen concentration all influence the in vivo activity. To systematically examine these factors, we selected ferulate 5-hydroxylase (F5H), a plant P450, with the Saccharomyces cerevisiae WAT11 strain as an expression host. Two media compositions and two cultivation procedures were investigated to optimize the in vivo activity of F5H. We modified a previously published selective growth medium (Pompon et al. [1996] Methods Enzymol 272:51-64) that increased the specific growth rate and cell yield of the host strain. A cultivation procedure with separate growth and induction stages that each contained selective media resulted in a 45% increase of whole cell F5H specific activity. In a medium designed for simultaneous growth and induction, we observed a 2.6-fold higher specific F5H activity, but substantially lower cell yield. Surprisingly, in this medium the higher specific F5H activity did not correlate with a higher P450 concentration. The effects of addition of the first committed heme precursor, delta-aminolevulinic acid, and Fe(III) at the beginning of induction period were also studied for our two-stage procedure. A small, but significant (P < 0.05) increase in whole cell F5H activity was observed following ALA addition.     

Sequence-specific DNA damage induced by carcinogenic danthron and anthraquinone in the presence of Cu(II), cytochrome P450 reductase and NADPH

The mechanism of metal-mediated DNA damage by carcinogenic danthron (1,8-dihydroxyanthraquinone) and anthraquinone was investigated by the DNA sequencing technique using ³²P-labeled human DNA fragments obtained from the human c-Ha-ras-1 proto-oncogene and the p53 tumor suppressor gene. Danthron caused DNA damage particularly at guanines in the 5'-GG-3', 5-GGGG-3', 5'-GGGGG-3' sequences (damaged bases are underlined) in the presence of Cu(II), cytochrome P450 reductase and the NADPH-generating system. The DNA damage was inhibited by catalase and bathocuproine, suggesting the involvement of H₂O₂ and Cu(I). The formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine increased with increasing concentration of danthron. On the other hand, carcinogenic anthraquinone induced less oxidative DNA damage than danthron. Electron spin resonance study showed that the semiquinone radical could beproduced by P450 reductase plus NADPH-mediated reduction of danthron, while little signal was observed with anthraquinone. These results suggest that danthron is much more likely to be reduced by P450 reductase and generate reactive oxygen species through the redox cycle, leading to more extensive Cu(II)-mediated DNA damage than anthraquinone. In the case of anthraquinone, its hydroxylated metabolites with similar reactivity to danthron may participate in DNA damage in vivo. We conclude that oxidative DNA damage by danthron and anthraquinone seems to be relevant for the expression of their carcinogenicity.     

Role of cytochrome P4501A1 in biotransformation of a tissue specific sarcomagen N-methyldibenzo[c,g]carbazole

7H-dibenzo[c,g]carbazole (DBC) is a potent liver and skin carcinogen, while its synthetic methyl derivative N-methyldibenzo[c,g]carbazole (MeDBC) is tissue specific sarcomagen. It is supposed that sarcomagenic activity of DBC depends on biotransformation at ring-carbon atoms, as with PAH, whereas the heterocyclic nitrogen plays an important role in liver carcinogenicity. The objective of this study was to elucidate the role of cytochrome P4501A1 in metabolic activation of sarcomagenic derivatives of DBC and to characterize the DNA damage profiles induced by DBC and MeDBC in relation to the mode of metabolic activation. The genetically engineered V79MZh1A1 cell line with stable expression of cDNA of human cytochrome P4501A1, the parental V79MZ cell line lacking any cytochrome P450 activity and human hepatocarcinoma Hep G2 cells were used as a model cells. Dose-dependent decrease in colony forming ability (CFA) was found in the V79MZh1A1 cell line after treatment of cells with DBC and MeDBC; however, no change in CFA was induced in parental V79MZ cells. These results were in a good correlation with DNA damaging effects of these two derivatives measured by the alkaline DNA unwinding (ADU) and the modified single cell gel electrophoresis (SCGE) techniques. Differences in DNA damage profiles induced by DBC and MeDBC were found in V79MZh1A1 and Hep G2 cells. These differences were probably the result of different reactive metabolite formation depending on chemical structure of the molecule and ways of biotransformation. This study showed that the cytochrome P4501A1 took part in activation of sarcomagenic DBC derivatives. Moreover, V79 cell lines with stable expression of different cytochromes P450 in combination with DNA repair endonucleases should be a useful tool for characterization of the role of individual cytochromes in metabolic activation pathways of DBC and MeDBC.     

Genotoxicity of Nicotiana tabacum leaves on Helix aspersa

Tobacco farmers are routinely exposed to complex mixtures of inorganic and organic chemicals present in tobacco leaves. In this study, we examined the genotoxicity of tobacco leaves in the snail Helix aspersa as a measure of the risk to human health. DNA damage was evaluated using the micronucleus test and the Comet assay and the concentration of cytochrome P450 enzymes was estimated. Two groups of snails were studied: one fed on tobacco leaves and one fed on lettuce (Lactuca sativa L) leaves (control group). All of the snails received leaves (tobacco and lettuce leaves were the only food provided) and water ad libitum. Hemolymph cells were collected after 0, 24, 48 and 72 h. The Comet assay and micronucleus test showed that exposure to tobacco leaves for different periods of time caused significant DNA damage. Inhibition of cytochrome P450 enzymes occurred only in the tobacco group. Chemical analysis indicated the presence of the alkaloid nicotine, coumarins, saponins, flavonoids and various metals. These results show that tobacco leaves are genotoxic in H. aspersa and inhibit cytochrome P450 activity, probably through the action of the complex chemical mixture present in the plant.     

热带假丝酵母转化烷烃过程中P450酶活的研究

a-、ω-长链二元酸(α-、ω-Long Chin Dicarboxylic Acid,DCA)是一种重要的化工原料,是合成工程塑料、香料、耐寒性增塑剂、涂料和液晶等物质的主要原料.目前,人们主要通过热带假丝酵母(Candidatropicalis)代谢烷烃来生产从DCA11到DCA18等不同碳链长的二元酸[1,2].多年来在各种微生物,尤其是假丝酵母的烷烃氧化途径方面有大量的研究[3,4].在假丝酵母转化烷烃生成长链二元酸的代谢过程中[5-7],烷烃被吸引进入细胞后,首先经过细胞色素P450酶(Cy-tochrome P450)氧化生成a-一元醇,再进一步被氧化生成a-一元酸,引过程称为a-氧化.     

Generation of Reactive Oxygen Species by Polyenylpyrroles Derivatives Causes DNA Damage Leading to G2/M Arrest and Apoptosis in Human Oral Squamous Cell Carcinoma Cells

Oral squamous cell carcinoma (OSCC) accounts for 5.8% of all malignancies in Taiwan and the incidence of OSCC is on the rise. OSCC is also a common malignancy worldwide and the five-year survival rate remains poor. Therefore, new and effective treatments are needed to control OSCC. In the present study we have investigated the efficacy and associated mechanisms of polyenylpyrroles and their analogs in both in vitro cell culture and in vivo nude mice xenografts. Auxarconjugatin B (compound 1a) resulted in cell cycle arrest in the G2/M phase and caspase-dependent apoptosis in OEC-M1 and HSC-3 cells by activating DNA damage and mitochondria dysfunction through the loss of mitochondrial membrane potential, release of cytochrome c, increase in B-cell lymphoma-2-associated X protein level, and decrease in B-cell lymphoma-2 level. Compound 1a-induced generation of intracellular reactive oxygen species through cytochrome P450 1A1 was identified as a major mechanism of its effect for DNA damage, mitochondria dysfunction and apoptosis, which was reversed by antioxidant N-acetylcysteine as well as cytochrome P450 1A1 inhibitor and specific siRNA. Furthermore, compound 1a-treated nude mice showed a reduction in the OEC-M1 xenograft tumor growth and an increase in the caspase-3 activation in xenograft tissue. These results provide promising insights as to how compound 1a mediates cytotoxicity and may prove to be a molecular rationale for its translation into a potential therapeutic against OSCC.     

Changes in the amount of cytochrome P450s in rat hepatic microsomes with starvation

The effects of starvation on the composition of 12 different cytochrome P450s in rat hepatic microsomes were studied with a specific antibody. Changes in the metabolic activity of the microsomes were studied at the same time. P450 DM (P450j) was induced 2.5-fold by a 48-h starvation and its increase reflected the increase of metabolic activity of hepatic microsomes toward aniline, 7-ethoxycoumarin, and N-nitrosodimethylamine. P450 K-5, the major renal cytochrome P450 in untreated male rat, was also induced 2.5-fold by a 48-h starvation. P450 UT-2 (P450h) and P450 UT-5 (P450g), typical male-specific forms, decreased with starvation. P450 UT-2 had high testosterone 2 alpha- and 16 alpha-hydroxylation activities. These activities of hepatic microsomes were reduced with the decrease in P450 UT-2. P450 PB-1, testosterone 6 beta-hydroxylase, was increased time-dependently by starvation. P450 UT-4 (RLM2), a minor male-specific form, was not changed by starvation. P450 PB-2 (P450k), present in both sexes, was changed little by starvation. P450 PB-4 (P450b) and P450 PB-5 (P450e) are strongly induced in rat liver by phenobarbital in coordinate fashion. Starvation increased P450 PB-4 12-fold but reduced P450 PB-5 to 22% of the control level. P450 MC-1 (P450d) was decreased by starvation. P450 MC-5 (P450c) was barely detected in control rats and was not changed by starvation. P450 IF-3 (P450a), rich in immature rats, was increased by starvation, accompanied by an increase in testosterone 7 alpha-hydroxylation activity in the hepatic microsomes. We further investigated whether new cytochrome P450s appeared upon starvation by comparison of chromatographic profiles of cytochrome P450 from starved rats with those of cytochrome P450 from control rats using HPLC. Three new cytochrome P450s were detected in the starved rats. These cytochrome P450s were purified to homogeneity. One of them was P450 DM, judging from spectral properties, catalytic activity, and the NH2-terminal sequence. The two other forms were designated P450 3b and 4b. The minimum molecular weights of P450 3b and 4b were 53,000 and 52,000, respectively, and their CO-reduced absorption maxima were at 449 and 452 nm, respectively. P450 3b metabolized aminopyrine, N-nitrosodimethylamine, 7-ethoxycoumarin, and lauric acid, but with low activity. P450 4b was efficient in lauric acid omega- and (omega-1)-hydroxylation only. The spectral properties, catalytic activity, peptide map, and NH2-terminal sequence of P450 4b agreed with those of P450 K-5. P450 3b was a new cytochrome P450, judged by these criteria.     

High sensitivity to 5-azacytidine in LEC rats, a strain with a metabolic predisposition to hepatitis and hepatoma: possible involvement of DNA methylation in the expression of cytochrome P-450 and gamma-glutamyl transpeptidase.

The 5-methylcytosine (5-mCyt) content in hepatic DNA of LEC rats was measured in order to know the mechanism by which changes in the cytochrome P-450 content and gamma-glutamyl transpeptidase activity occur. At the age of 10 or 16 weeks, there was no difference in the extent of DNA methylation as compared with that of control strain (LEA) rats. However, in the hepatoma tissues that developed later in LEC animals, the percentage of 5-mCyt in the liver of LEC rats was markedly reduced. A single i.p. dose of 5-azacytidine brought about a significant reduction of 5-mCyt content with a concomitant decrease of cytochrome P-450 and an increase in gamma-glutamyl transpeptidase activity in LEC rats, whereas no such changes occurred in the control LEA rats. These results suggest that LEC rats are highly sensitive to 5-azacytidine and that a reduction in hepatic DNA methylation may play some role in the predisposition of the rats to hepatitis or hepatoma.     

Acetaldehyde activation of poly(ADP-ribose)polymerase in hepatocytes of mice treated in vivo

The activities of poly(ADP-ribose) polymerase and of DNA polymerases alpha and beta and the level of cytochrome P450 were determined in mouse parenchymal liver cells 5 h after treatment with 0.1, 0.3, 1.0, and 3.0 mumole of acetaldehyde. Injection with 1.0 and 3.0 mumole of acetaldehyde induced an increase in poly(ADP-ribose) polymerase activity and in the P450 level, but had no effect on DNA polymerases. The stimulation of poly(ADP-ribose) polymerase activity can be used as an index of induced DNA damage. The possibility of using this experimental approach with other cells derived from mice treated in vivo with different xenobiotics is discussed.     

Copper-mediated oxidative DNA damage induced by eugenol: possible involvement of O-demethylation

Eugenol used as a flavor has potential carcinogenicity. DNA adduct formation via 2,3-epoxidation pathway has been thought to be a major mechanism of DNA damage by carcinogenic allylbenzene analogs including eugenol. We examined whether eugenol can induce oxidative DNA damage in the presence of cytochrome P450 using [32P]-5'-end-labeled DNA fragments obtained from human genes relevant to cancer. Eugenol induced Cu(II)-mediated DNA damage in the presence of cytochrome P450 (CYP)1A1, 1A2, 2C9, 2D6, or 2E1. CYP2D6 mediated eugenol-dependent DNA damage most efficiently. Piperidine and formamidopyrimidine-DNA glycosylase treatment induced cleavage sites mainly at T and G residues of the 5'-TG-3' sequence, respectively. Interestingly, CYP2D6-treated eugenol strongly damaged C and G of the 5'-ACG-3' sequence complementary to codon 273 of the p53 gene. These results suggest that CYP2D6-treated eugenol can cause double base lesions. DNA damage was inhibited by both catalase and bathocuproine, suggesting that H2O2 and Cu(I) are involved. These results suggest that Cu(I)-hydroperoxo complex is primary reactive species causing DNA damage. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine was significantly increased by CYP2D6-treated eugenol in the presence of Cu(II). Time-of-flight-mass spectrometry demonstrated that CYP2D6 catalyzed O-demethylation of eugenol to produce hydroxychavicol, capable of causing DNA damage. Therefore, it is concluded that eugenol may express carcinogenicity through oxidative DNA damage by its metabolite.