Induction Of CYP2A5 Research Articles

Nrf2-mediated induction of Cyp2a5 partially protects against reductive endoplasmic reticulum stress in mouse hepatocytes

Cytochrome P450 2a5 (Cyp2a5) is distinct from other P450 enzymes in that it is induced in the endoplasmic reticulum (ER) of mouse hepatocytes in conditions that are injurious to the liver. These conditions cause ER stress eventually resulting in apoptosis if not rectified. We previously showed that mouse hepatic Cyp2a5 is induced during reductive ER stress caused by the intramolecular disulfide form of dithiothreitol, trans-4,5-dihydroxy-1,2-dithiane (DTTox), and that overexpression of Cyp2a5 provides partial protection against apoptosis due to bilirubin (BR), a compound known to cause ER stress. The purpose of this study was to investigate the mechanism of Cyp2a5 gene regulation by DTTox and to determine if Cyp2a5 plays a cytoprotective role during reductive ER stress. Exposure to DTTox (10 mM) and another reductive ER stressor, 2-mercaptoethanol (1 mM), for 48 h markedly increased Cyp2a5 protein levels in primary mouse hepatocytes. In addition, DTTox transactivated Cyp2a5 via a mechanism involving the transcription factor nuclear factor-(erythroid-derived 2)-like 2 (Nrf2). Expression of the BR-conjugating enzyme, UDP glucuronosyl transferase 1A1 (UGT1A1) was also increased after DTTox treatment, however, this was reduced by Cyp2a5 overexpression. Hemin, a porphyrin inducer of Cyp2a5, induced mRNA splicing of X-box binding protein 1 (XBP-1), a transcription factor involved in the ER stress response, however, this was also reduced by Cyp2a5 overexpression. Finally, overexpression of Cyp2a5 partially blocked DTTox-mediated caspase-3 cleavage in Hepa 1–6 cells suggesting a cytoprotective role during ER stress. These findings demonstrate that Nrf2-mediated induction of Cyp2a5 in a reducing ER environment provides partial protection against ER stress-induced apoptosis by decreasing XBP-1 mRNA splicing and caspase-3 cleavage.

Cytochrome P450s and Alcoholic Liver Disease.

Alcohol consumption causes liver diseases, designated as Alcoholic Liver Disease (ALD). Because alcohol is detoxified by alcohol dehydrogenase (ADH), a major ethanol metabolism system, the development of ALD was initially believed to be due to malnutrition caused by alcohol metabolism in liver. The discovery of the microsomal ethanol oxidizing system (MEOS) changed this dogma. Cytochrome P450 enzymes (CYP) constitute the major components of MEOS. Cytochrome P450 2E1 (CYP2E1) in MEOS is one of the major ROS generators in liver and is considered to be contributive to ALD. Our labs have been studying the relationship between CYP2E1 and ALD for many years. Recently, we found that human CYP2A6 and its mouse analog CYP2A5 are also induced by alcohol. In mice, the alcohol induction of CYP2A5 is CYP2E1-dependent. Unlike CYP2E1, CYP2A5 protects against the development of ALD. The relationship of CYP2E1, CYP2A5, and ALD is a major focus of this review.

Alcoholic Liver Disease: from CYP2E1 to CYP2A5.

This article reviews recent studies on CYP2E1-mediated alcoholic liver injury, the induction of CYP2A5 by alcohol and the mechanism for this upregulation, especially the permissive role of CYP2E1 in the induction of CYP2A5 by alcohol and the CYP2E1-ROS-Nrf2 pathway, and protective effects of CYP2A5 against ethanol-induced oxidative liver injury. Ethanol can induce CYP2E1, an active generator of reactive oxygen species (ROS), and CYP2E1 is a contributing factor for alcoholinduced oxidative liver injury. CYP2A5, another isoform of cytochrome P450, can also be induced by ethanol. Chronic feeding of ethanol to wild type mice increased CYP2A5 catalytic activity, protein and mRNA levels as compared to pair-fed controls. This induction was blunted in CYP2E1 knockout (cyp2e1-/-) mice but was restored when human CYP2E1 was reintroduced and expressed in cyp2e1-/- mice. Ethanol-induced CYP2E1 co-localized with CYP2A5 and preceded the elevation of CYP2A5. The antioxidants N-acetyl cysteine and vitamin C lowered the alcohol elevation of ROS and blunted the alcohol induction of CYP2A5, but not CYP2E1, suggesting ROS play a novel role in the crosstalk between CYP2E1 and CYP2A5. The antioxidants blocked the activation of Nrf2, a transcription factor known to upregulate expression of CYP2A5. When alcohol-induced liver injury was enhanced in Nrf2 knockout (Nrf2-/-) mice, alcohol elevation of CYP2A5 but not CYP2E1 was also lower in Nrf2-/- mice. CYP2A5 knockout (cyp2a5-/-) mice exhibited an enhanced alcoholic liver injury compared with WT mice as indicated by serum ALT, steatosis and necroinflammation. Alcohol-induced hyperglycemia were observed in cyp2a5-/- mice but not in WT mice.

Possible involvement of nuclear factor erythroid 2-related factor 2 in the gene expression of Cyp2b10 and Cyp2a5

Cytochrome P450 gene expression is altered by various chemical compounds. In this study, we used nuclear factor erythroid 2-related factor 2 (Nrf2)–deficient (Nrf2−⧸−) mice to investigate the involvement of Nrf2 in Cyp2b10 and Cyp2a5 gene expression. Phorone, an Nrf2 activator, strongly increased Cyp2b10 and Cyp2a5 mRNA as well as Nrf2 target genes, including NAD(P)H-quinone oxidoreductase-1 and heme oxygenase-1, in wild-type mouse livers 8h after treatment. The phorone-induced mRNA levels in Nrf2−⧸− mouse livers were lower than that in wild-type mouse livers. Nrf2−⧸− mice showed attenuated Cyp2b10 and Cyp2a5 induction by phenobarbital, a classical Cyp2b inducer. These findings suggest that the Nrf2 pathway is involved in Cyp2b10 and Cyp2a5 gene expression.

Ethanol Induction of CYP2A5: Role of CYP2E1-ROS-Nrf2 Pathway

Chronic ethanol consumption was previously shown to induce CYP2A5 in mice, and this induction of CYP2A5 by ethanol was CYP2E1 dependent. In this study, the mechanisms of CYP2E1-dependent ethanol induction of CYP2A5 were investigated. CYP2E1 was induced by chronic ethanol consumption to the same degree in wild-type (WT) mice and CYP2A5 knockout (Cyp2a5 (-/-)) mice, suggesting that unlike the CYP2E1-dependent ethanol induction of CYP2A5, ethanol induction of CYP2E1 is not CYP2A5 dependent. Microsomal ethanol oxidation was about 25% lower in Cyp2a5 (-/-) mice compared with that in WT mice, suggesting that CYP2A5 can oxidize ethanol although to a lesser extent than CYP2E1 does. CYP2A5 was induced by short-term ethanol consumption in human CYP2E1 transgenic knockin (Cyp2e1 (-/-) KI) mice but not in CYP2E1 knockout (Cyp2e1 (-/-)) mice. The redox-sensitive transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) was also induced by acute ethanol in Cyp2e1 (-/-) KI mice but not in Cyp2e1 (-/-) mice. Ethanol induction of CYP2A5 in Nrf2 knockout (Nrf2 (-/-)) mice was lower compared with that in WT mice, whereas CYP2E1 induction by ethanol was comparable in WT and Nrf2 (-/-) mice. Antioxidants (N-acetyl-cysteine and vitamin C), which blocked oxidative stress induced by chronic ethanol in WT mice and acute ethanol in Cyp2e1 (-/-) KI mice, also blunted the induction of CYP2A5 and Nrf2 by ethanol but not the induction of CYP2E1 by ethanol. These results suggest that oxidative stress induced by ethanol via induction of CYP2E1 upregulates Nrf2 activity, which in turn regulates ethanol induction of CYP2A5. Results obtained from primary hepatocytes, mice gavaged with binge ethanol or fed chronic ethanol, show that Nrf2-regulated ethanol induction of CYP2A5 protects against ethanol-induced steatosis.

CYP2A5 Induction and Hepatocellular Stress: An Adaptive Response to Perturbations of Heme Homeostasis

Unlike most cytochrome P450 (CYP) enzymes, murine hepatic CYP2A5 is induced during pathological conditions that result in liver injury including hepatotoxicity mediated by xenobiotics, hepatitis caused by various microbial agents and liver neoplasia. Since CYP2A5 metabolizes various important xenobiotics including nicotine and pro-carcinogens such as nitrosamines and aflatoxin B(1), altered gene expression could affect tobacco addiction, hepatotoxicity and hepatocarcinogenesis. This article synthesizes the current knowledge concerning hepatic expression of Cyp2a5 including the transcriptional and post-transcriptional regulatory mechanisms, pathophysiological conditions associated with enzyme induction such as oxidative and endoplasmic reticulum stress and altered lipid and energy homeostasis as well as the known exogenous and putative endogenous substrates. Knowledge of the stimuli responsible for the unique overexpression of CYP2A5 during liver injury may provide clues to a functional role for this enzyme and the impact of variable CYP2A5 expression on xenobiotic metabolism and toxicity, disease development and the adaptive response to hepatocellular stress.

Ethanol Induction of CYP2A5: Permissive Role for CYP2E1

CYP2A5 metabolizes xenobiotics and activates hepatocarcinogens, and induction occurs in response to hepatic damage and cellular stress. We evaluated whether ethanol can elevate CYP2A5 and whether CYP2E1 plays a role in the ethanol induction of CYP2A5. Wild-type (WT), CYP2E1 knockout (KO), and CYP2E1 knockin (KI) mice were fed ethanol for 3 weeks. Ethanol increased CYP2E1 and CYP2A5 protein and activity in WT mice but not in the KO mice. Induction of CYP2A5 (and CYP2E1) was restored in the KI mice. Ethanol induction of CYP2A5 occurred only after CYP2E1 was first induced. Immunohistochemical staining revealed that CYP2E1 and CYP2A5 colocalize to the same zones in the liver. Ethanol also elevated CYP2A5 mRNA levels in WT and KI mice but not in KO mice. Induction of CYP2A5 by cadmium was partially decreased in KO mice compared with WT or KI mice. Ethanol elevated CYP2A4 mRNA levels in all mice although the extent of induction was lowest in the KO mice. In summary, ethanol elevated mouse hepatic CYP2A5 levels, which may be of toxicological significance because CYP2A5 metabolizes nicotine and other drugs and activates hepatocarcinogens. Induction of CYP2A5 by ethanol is potentiated by the induction of CYP2E1. We speculate that ethanol induction of CYP2E1 followed by increases in reactive oxygen species and activation of Nrf2 are important steps in the mechanism by which ethanol induces CYP2A5. The possibility that induction of CYP2E1 is permissive for the induction of CYP2A5 may reflect a new contribution by CYP2E1 to the actions of ethanol.

Up- and down-modulation of liver cytochrome P450 activities and associated events in two murine malaria models

BackgroundThe mechanisms by which malaria up and down-regulates CYP activities are not understood yet. It is also unclear whether CYP activities are modulated during non-lethal malaria infections. This study was undertaken to evaluate the time course of CYP alterations in lethal (Plasmodium berghei ANKA) and non-lethal (Plasmodium chabaudi chabaudi) murine malaria. Additionally, hypotheses on the association of CYP depression with enhanced nitric oxide (NO) production, and of CYP2a5 induction with endoplasmic reticulum dysfunction, enhanced haem metabolism and oxidative stress were examined as well.MethodsFemale DBA-2 and C57BL/6 mice were infected with P.berghei ANKA or P. chabaudi and killed at different post-infection days. Infection was monitored by parasitaemia rates and clinical signs. NO levels were measured in the serum. Activities of CYP1a (ethoxyresorufin-O-deethylase), 2b (benzyloxyresorufin-O-debenzylase), 2a5 (coumarin-7-hydroxylase) and uridine-diphosphoglucuronyl-transferase (UGT) were determined in liver microsomes. Glutathione-S-transferase (GST) activity and concentrations of gluthatione (GSH) and thiobarbituric acid-reactive substances (TBARS) were determined in the liver. Levels of glucose-regulated protein 78 (GRP78) were evaluated by immunoblotting, while mRNAs of haemoxygenase-1 (HO-1) and inducible nitric oxide synthase (iNOS) were determined by quantitative RT-PCR.ResultsPlasmodium berghei depressed CYP1a and 2b and induced 2a5 in DBA-2 mice. In P.berghei-infected C57BL/6 mice CYP activities remained unaltered. In both strains, GST and UGT were not affected by P.berghei. Plasmodium c. chabaudi depressed CYP1a and 2b and induced 2a5 activities on the day of peak parasitaemia or near this day. CYP2a5 induction was associated with over-expression of HO-1 and enhanced oxidative stress, but it was not associated with GRP78 induction, a marker of endoplasmic reticulum stress. Plasmodium chabaudi increased serum NO on days near the parasitaemia peak in both strains. Although not elevating serum NO, P.berghei enhanced iNOS mRNA expression in the liver.ConclusionDown-regulation of CYP1a and 2b and induction of 2a5 occurred in lethal and non-lethal infections when parasitaemia rates were high. A contribution of NO for depression of CYP2b cannot be ruled out. Results were consistent with the view that CYP2a5 and HO-1 are concurrently up-regulated and suggested that CYP2a5 induction may occur in the absence of enhanced endoplasmic reticulum stress.

Microarray analysis of hepatic gene expression in pyrazole-mediated hepatotoxicity: Identification of potential stimuli of Cyp2a5 induction

Cytochrome P450 2a5 ( Cyp2a5) expression is induced during liver damage caused by hepatotoxins such as pyrazole, however, the mechanism underlying this overexpression is unclear. In order to identify pathophysiological and cellular responses to pyrazole that might alter Cyp2a5 expression, we examined the effect of pyrazole on mouse hepatic gene expression in C57BL/6 mice using Affymetrix 430 2.0 microarrays. Over 3000 differentially expressed genes were identified 24-h after pyrazole treatment that were associated with a variety of cellular pathways. Upregulated genes were primarily involved in the splicing and processing of RNA and the unfolded protein response pathway, while downregulated genes were associated with amino acid and lipid metabolism, and generation of precursor metabolites for energy production. We also examined the effects of pyrazole on cellular pathways linked to metabolic and histopathological changes observed with pyrazole toxicity. Increased mRNA levels were observed for genes involved in bilirubin production, whereas the major genes of the urea cycle were strongly decreased. Changes in genes involved in carbohydrate metabolism were also observed which could explain pyrazole-induced glycogen depletion and decreased serum glucose. In addition, over 100 genes involved in the cellular stress response were upregulated by pyrazole treatment, including genes involved in the unfolded protein response and redox status. Based on these results and previous evidence concerning the regulation of Cyp2a5, we have identified several pathophysiological changes including altered energy homeostasis, hyperbilirubinemia, ER stress, and altered redox status that are associated with CYP2A5 overexpression and may represent potential stimuli for the induction of Cyp2a5.

Plasmodium berghei (ANKA): Infection induces CYP2A5 and 2E1 while depressing other CYP isoforms in the mouse liver

It has been reported that malaria infection impairs hepatic drug clearance and causes a down-regulation of CYP-mediated monooxygenase activities in rodents and humans. In the present study, we investigated the effects of Plasmodium berghei infection on the activity of liver monooxygenases in female DBA/2 and C57BL/6 mice. In both mouse strains, P. berghei infection decreased activities mediated by CYP1A (EROD: DBA/2 65.3%, C57BL/6 44.7%) and 2B (BROD: DBA/2 64.3%, C57BL/6 49.8%) subfamily isoforms and increased activities mediated by 2A5 (COH: DBA/2 182.4%, C57BL/6 148.5%) and 2E1 (PNPH: DBA/2 177.8%, C57BL/6 128.5%) isoforms as compared to non-infected controls. Since malaria infection also produced an increase in ALT (273.1%) and AST (354.1%) activities in the blood serum, our findings are consistent with the view that CYP2A5 activity is induced by liver injury. An almost generalized depression of CYP-mediated activities has been found with numerous infections and inflammatory stimuli but an induction of CYP2A5 had been previously noted only in some viral hepatitis and trematode (liver fluke) infections.

Alcohol Upregulation of CYP2A5: Role of Reactive Oxygen Species

Hepatic cytochrome P450 (CYP) 2E1 and CYP2A5 activate many important drugs and hepatotoxins. CYP2E1 is induced by alcohol, but whether CYP2A5 is upregulated by alcohol is not known. This article reviews recent studies on the induction of CYP2A5 by alcohol and the mechanism and role of reactive oxygen species (ROS) in this upregulation. Chronic feeding of ethanol to wild type mice increased CYP2A5 catalytic activity and protein and mRNA levels. This induction was blunted in CYP2E1 knockout mice and by a CYP2E1 inhibitor, but was restored in CYP2E1 knockin mice, suggesting a role for CYP2E1 in the induction of CYP2A5 by alcohol. Since CYP2E1 actively generates ROS, the possible role of ROS in the induction of CYP2A5 by alcohol was determined. ROS production was elevated by ethanol treatment. The antioxidants N-acetyl cysteine and vitamin C lowered the alcohol-induced elevation of ROS and blunted the alcohol-mediated induction of CYP2A5. These results suggest that ROS play a novel role in the crosstalk between CYP2E1 and CYP2A5. Alcohol treatment activated nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2), a transcription factor which up-regulates expression of CYP2A5. The antioxidants blocked the activation of Nrf2. The alcohol-induced elevation of CYP2A5, but not CYP2E1, was lower in Nrf2 knockout mice. We propose that increased generation of ROS from the alcohol-induced CYP2E1 activates Nrf2, which subsequently up-regulates the expression of CYP2A5. Thus, a novel consequence of the alcohol-mediated induction of CYP2E1 and increase in ROS is the activation of redox-sensitive transcription factors, such as Nrf2, and expression of CYP2A5. Further perspectives on this alcohol-CYP2E1-ROS-Nrf2-CYP2A5 pathway are presented.

Regulation of theCyp2a5Gene Involves an Aryl Hydrocarbon Receptor-Dependent Pathway

We have investigated the role of the aryl hydrocarbon receptor (AHR) in the regulation of the Cyp2a5 gene. The C57BL/6 and DBA/2 mouse strains with a genetically determined difference in AHR function were used to study the CYP2A5 induction by typical AHR ligands, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene. The CYP2A5 mRNA up-regulation in these mouse strains showed a difference in response, typical for AHR-regulated genes, both by TCDD in cultured primary hepatocytes and by 3-methylcholanthrene in vivo. In primary hepatocytes, TCDD caused a 3-fold elevation of the CYP2A5 protein level and a similar induction of the CYP2A5-catalyzed coumarin 7-hydroxylation activity. In reporter gene assays, the Cyp2a5 promoter region -3033 to +10 mediated a 2- to 5-fold induction of luciferase activity by TCDD treatment in primary hepatocytes and in Hepa-1 hepatoma cells with an intact AHR/AHR nuclear translocator (ARNT) complex. In Hepa-1 variant cell lines with deficiencies in the AHR/ARNT complex, the absence of ARNT abolished the induction. A putative AHR response element (XRE) was identified in the Cyp2a5 promoter at the position -2514 to -2492 and found to interact with the AHR/ARNT heterodimer. Transfection experiments combined with mutation of the XRE site indicated that the site partly mediates the TCDD induction of Cyp2a5. An additional AHR-dependent mechanism also regulates the proximal promoter of the Cyp2a5 gene. In conclusion, our studies showed that AHR ligands up-regulate Cyp2a5 transcriptionally by an AHR/ARNT-dependent mechanism and established Cyp2a5 as a novel AHR-regulated gene.

Effects of lipopolysaccharide-stimulated inflammation and pyrazole-mediated hepatocellular injury on mouse hepatic Cyp2a5 expression

Murine hepatic cytochrome P450 2a5 (Cyp2a5) is induced during hepatotoxicity and hepatitis, however, the specific regulatory mechanisms have not been determined. We compared the influence of acute inflammation elicited in vivo by bacterial endotoxin lipopolysaccharide (LPS) and liver injury caused by the hepatotoxin pyrazole on hepatic Cyp2a5 expression in mice. Pyrazole treatment resulted in statistically significant increases in levels of Cyp2a5 mRNA, protein and catalytic activity by 540, 273 and 711%, respectively ( P<0.05). In LPS-treated livers Cyp2a5 expression was significantly reduced compared to controls at the mRNA (46%) protein (35%), and activity (23%) levels ( P<0.05). Treatment of mice with recombinant murine interleukin-1 beta and interleukin-6 had no significant effect on Cyp2a5 mRNA and protein levels. Liver injury, as assessed by serum alanine aminotransferase, was greater with pyrazole than with LPS treatment (609 vs 354% of control levels respectively). ER stress, determined by hepatic glucose regulated protein 78 (grp78) levels, was greater with pyrazole (185% of controls) than with LPS (128% of controls). In pyrazole-treated liver, overexpression of immunoreactive grp78 protein revealed that ER stress was localized to pericentral hepatocytes in which Cyp2a5 was induced. Evidence of glycogen loss and membrane damage in these cells was suggestive of oxidative damage. Moreover, vitamin E attenuated Cyp2a5 induction by pyrazole in vivo. These results suggest that induction of Cyp2a5 that has been observed in mouse models of hepatitis and hepatoxicity may be related to oxidative injury to the endoplasmic reticulum of pericentral hepatocytes rather than exposure to pro-inflammatory cytokines.

Acute sodium arsenite treatment induces Cyp2a5 but not Cyp1a1 in the C57Bl/6 mouse in a tissue (kidney) selective manner

Modulation of hepatic and extrahepatic detoxication enzymes Cyp1a1, Cyp2a5, glutathione S-transferse Ya (GSTYa) and NAD(P)H:quinone oxidoreductase (QOR) dependent catalytic activity and mRNA levels were investigated at 1, 2, or 4 days in liver, lung, or kidney of male, adult CD57 Bl/6 mice treated sc with a single dose (85 micromol/kg) of sodium arsenite (As3+). Maximum decreases of total hepatic cytochrome P450 (CYP) monooxygenase content and catalytic activities, occurring at 24 h, corresponded with maximum increases of heme oxygenase (HO-1) in all tissues, as well as maximum plasma total bilirubin. Extrahepatic increases in CYP were observed only in non-AHR dependent isozymes in the kidney, where both Cyp2a5 mRNA and catalytic activity increased maximally 24 h after treatment. In contrast, no significant changes in Cyp2b1/2-dependent PROD or mRNA activity and decreases in Cyp1a1-dependent-EROD activity were noted 1, 2, or 4 days after treatment. Increases in QOR catalytic activities were observed in all tissues examined with increased mRNA in kidney. On the other hand, GSTYa catalytic activity and mRNA increases were only detected in kidney. This study demonstrates the differential modulation of CYP, QOR, and GST-Ya, important drug metabolizing enzymes after acute As3+ administration. The induction of Cyp2a5, QOR, and GSTYa catalytic activity and gene expression occurred primarily in kidney during or shortly after conditions of oxidant stress.

Induction of CYP2A5 by pyrazole and its derivatives in mouse primary hepatocytes.

Mouse liver CYP2A5 is induced by several structurally unrelated compounds. In intact mouse liver, pyrazole (PYR) and 4-hydroxypyrazole (4-OH) induce selectively the expression of CYP2A5 while expression of other CYPs is decreased. In this study we exposed mouse primary hepatocytes to PYR, 4-OH, 4-methylpyrazole (4Me; 0.1-20 mM) and 4-iodopyrazole (4-I; 0.1-5.0 mM). PYR and its derivatives increased coumarin 7-hydroxylase activity, with 4-1 and 4-OH being the strongest inducers, by 114-fold and 41-fold, respectively. However, only 4-1 treatment increased markedly the CYP2A5 protein content. CYP2B9/10-mediated pentoxyresorufin O-deethylase activity (PROD) was decreased by 80% by 4-Me and 4-1, and by 50% by 4-OH while PYR had no marked effect. PYR and 4-Me increased 2- to 3-fold the CYPA1/2-mediated ethoxyresorufin O-deethylase activity (EROD) while 4-OH and 4-1 had no marked effect on this enzyme. The time of exposure markedly affected the inducibility of 4-OH such that induction was 7-fold stronger when it was added to the incubation medium 24 h after the isolation of hepatocytes compared to exposure 3 h after their isolation. Cimetidine prevented the induction of coumarin 7-hydroxylase activity by PYR and 4-OH by 46 and 74%, respectively indicating that their effects on the expression of CYP2A5 are, at least partly, mediated via their metabolites. The data demonstrate that the regulation of CYP2A5 is different from other monooxygenases and that the effects of pyrazole and its derivatives are different in vivo and in vitro. Also, the timing of exposure markedly affects the inducibility of 4-OH in hepatocytes.

Regulation of CYP 2 A 5 induction by porphyrinogenic agents in mouse primary hepatocytes.

All cytochrome P450 (CYP) enzymes contain heme as a prosthetic group. In contrast to other CYP enzymes, murine CYP 2 A 5 is upregulated in vivo by several agents that disturb cellular heme balance. To test the hypothesis that porphyrinogenic agents have the common feature of being able to increase CYP 2 A 5 expression, mouse liver primary hepatocytes were exposed to various porphyrinogenic chemicals and changes in CYP 2 A 5 catalytic activity and levels of mRNA were monitored. Phenobarbital increased hepatic CYP 2 A 5-mediated coumarin 7-hydroxylase (COH) activity (13.2-fold) and the amount of CYP 2 A 5 steady-state mRNA (10.6-fold). Hepatocyte COH activity was increased also by the ferrochelatase inhibitor griseofulvin and the protoporphyrinogen oxidase inhibitor acifluorfen (about 9-fold induction). Of these inducers, only phenobarbital affected CYP 1 A 12 and CYP 2 B 10 expression. In contrast, many other porphyrinogenic agents such as cobalt, 2,2,4-trimethyl-1,2-dihydroquinoline (TMDQ), 1-[4-(3-acetyl-2,4,6-trimethylphenyl)-2,6-cyclohexanedionyl]-O-eth yl propionaldehyde oxime (ATMP), aminotriazole, and thioacetamide either decreased or had no effect on CYP 2 A 5. The increases in COH activity and CYP 2 A 5 mRNA were unaffected by combined treatment with the inducers and heme arginate, suggesting that heme is not a regulator of CYP 2 A 5 induction. Treatment with actinomycin D totally abolished both constitutive CYP 2 A 5 expression and its inducibility, suggesting that a transcriptional component is involved. These data suggest that, in mouse primary hepatocytes, CYP 2 A 5 induction is not a universal response to disturbed cellular heme biosynthesis.

Chemopreventive effects of fermented brown rice and rice bran against 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis in female A/J mice

The most common cause of human lung cancer is suggested to be exposure to the carcinogens in tobacco smoke. Among the multiple chemicals in tobacco smoke, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) has been regarded as one of the important agents for generation of lung cancers. Previously, our studies proved that fermented brown rice and rice bran (FBRA) has chemopreventive effects against carcinogenesis of the colon, liver, stomach, bladder and esophagus. In the present study, we examined possible chemopreventive effects of FBRA on the NNK-induced lung tumorigenesis in mice. Six-week-old female A/J mice were divided into 8 groups, and groups 1-5 were given NNK (10 mmol) by i.p. injection at week 7. Groups 2 and 3 were fed with diet containing 5 and 10% FBRA during the initiation phase, respectively. Groups 4 and 5 were fed with 5% and 10% FBRA during the post-initiation phase. Groups 1 and 6 were given control diet throughout the experiment. Groups 7 and 8 were given the diet containing 5 and 10% FBRA throughout the experiment, respectively. In both initiation (group 3) and post-initiation phase (group 5), 10% FBRA exposure significantly reduced the multiplicity of lung tumor (group 3, 2.35+/-2.13; group 5, 3.00+/-1.52; group 1, 4.08+/-1.85; p<0.006 and 0.04, respectively). Furthermore, administration of FBRA during the post-initiation phase significantly decreased the tumor size in comparison with that of control mice (0.66+/-0.32 vs. 0.77+/-0.33 mm). Treatment of 10% FBRA significantly reduced the mRNA expression levels of cytochrome P450 2A5 (Cyp2a5), which is known to be closely related to the human CYP2A6 enzyme that is involved in the mutagenic activation of NNK, in the lung but not liver tissues. A significantly reduced index of Ki67 positivity of lung tumors in group 5 was confirmed when compared with tumors of the control group (0.065+/-0.016 vs. 0.114+/-0.025). These findings suggest that FBRA has inhibitory effects on NNK-induced pulmonary tumorigenesis in A/J mice in both during initiation and post-initiation treatment, which is possibly associated with the induction of Cyp2a5 in the lung and the reduced proliferation rate of tumor cells. FBRA may be a promising chemopreventive agent for human lung cancers.