The Evolution of Drug Metabolism and Disposition: A Perspective From the Editors.

This article was solicited to commemorate the 50th anniversary of Drug Metabolism and Disposition (DMD) and features perspectives from five former editors spanning the years 1994 to 2020. During that time frame the journal underwent significant changes in manuscript submission and processing as well as multiple generational changes in the composition of the editorial board and associate editors. A constant, however, has been the commitment to be the premier journal for publications of articles in the areas of drug metabolism, absorption, distribution, excretion, and pharmacokinetics. Advances in some of those areas during the past 3 decades have been monumental. Two cases in point involve cytochromes P450 and drug transporters. In 1994 rigorous characterization of human cytochrome P450 enzymes was in its infancy, there were no proven selective inhibitors, and the idea of solving a human P450 X-ray crystal structure was just a fantasy. Likewise, little was known about individual drug transporters. Today, detailed knowledge of individual human P450 enzymes and drug transporters is integral in drug design and drug discovery and in avoiding drug interactions. In the face of these huge advances in knowledge, each editor has been charged with maintaining the caliber and significance of the journal and its financial solvency while serving the needs of individual authors. We present 5 individual perspectives on the challenges and rewards of serving as DMD editor and hope that, by humanizing the job, we will encourage others to assume positions of responsibility in publication of society journals. SIGNIFICANCE STATEMENT: The 5 most recent former editors of DMD describe their experiences and perspectives on the position in the context of constantly changing scientific emphases, technology, and publishing practices. The article offers subscribers, authors, and future editors and editorial board members valuable insights into the inner workings of the journal.

Insights into insulin-mediated regulation of CYP2E1: miR-132/-212 targeting of CYP2E1 and role of phosphatidylinositol 3-kinase, Akt (protein kinase B), mammalian target of rapamycin signaling in regulating miR-132/-212 and miR-122/-181a expression in primary cultured rat hepatocytes.

Several microRNAs (miRNAs) were selected for characterization of their response to insulin signaling based on in silico predictions of targeting CYP2E1 mRNA and previous reports implicating their role in hepatic metabolism and disease. CYP2E1 expression decreases with increasing insulin concentration and has been shown to be regulated by the phosphatidylinositol 3-kinase (PI3-K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway. In primary cultured rat hepatocytes, insulin at 0.1, 1.0, and 10 nM elevated miRNA-132 and -212 expression ∼2- and 1.8-fold, respectively, whereas expression of miRNA-181a and -122 increased ∼1.6- and 1.4-fold, respectively. In contrast, insulin failed to alter significantly the expression of miRNA let-7a. Mechanistic studies using inhibitors of PI3-K, Akt, and mTOR were used to examine the role of the insulin signaling pathway on miR expression and resulted in significant suppression of the insulin-mediated elevation of miR-132, miR-212, and miR-122 levels, with a lesser effect observed for miR-181a. Targeting of the rat CYP2E1 3'-untranslated region (UTR) by miR-132 and -212 was demonstrated with an in vitro luciferase reporter assay. These data show that insulin, which regulates CYP2E1 through the PI3-K, Akt, mTOR signaling pathway, also regulates the expression of miRs that target the 3'-UTR of CYP 2E1 mRNA and are involved in the regulation of hepatic metabolism and disease.

Proteomic and phosphoproteomic alterations in benign, premalignant and tumor human breast epithelial cells and xenograft lesions: biomarkers of progression.

The MCF10A human breast epithelial cell lineage includes the benign MCF10A cells, premalignant cells (MCF10AT, MCF10ATG3B) and malignant MCF10CA1a tumor cells. The premalignant and tumor cells recapitulate the progressive alterations associated with the temporal development of PBD and carcinoma. Ras protein levels were elevated by 6.9-, 22.4- and 32.2-fold in 10AT, 10ATG3B and 10CA1a cells, respectively, relative to 10A cells. K-Ras was not detected, N-Ras levels were unchanged; Rac and Rho levels increased in 10CA1a tumor cells. Phospho-phosphatidylinositol 3-kinase, phosphoinositide-dependent protein kinase 1 (PDK1), phospho-PDK1, phospho-eukaryotic translation initiation factor 4E (eIF4E) and phospho-eukaryotic initiation factor 4E binding protein 1 (4E-BP1) levels progressively increased in the cell lineage, with the greatest increase monitored in 10CA1a tumor cells. Phospho Ser 473 and Thr 408 Akt levels increased 10.2- and 136-fold in 10CA1a cells, respectively, relative to 10A cells. Phospho-p70S6 kinase (p70S6K) increased >2-fold in 10CA1a cells, relative to 10A cells. Immunohistochemistry confirmed Ras, phospho-Akt and phospho-p70S6K (Thr 421/ Ser 424) expression in lesions arising from premalignant and tumor cells. FOXO 1, phospho-FOXO 1 and phospho-FOXO 4 were significantly elevated in 10ATG3B premalignant and 10CA1a tumor cells. Phospho-FOXO 3a was progressively elevated, with the greatest levels detected in 10CA1a tumor cells. Immunohistochemistry revealed that phospho-FOXO 1, 3a and 4 staining was less in benign lesions, but elevated in advanced 10ATG3B and malignant 10CA1a lesions, showing a correspondence between the cells and lesions. Hence, phospho-Akt and phospho-FOXO 1, 3a and 4 merit consideration as biomarkers of tumorigenic risk from hyperplastic breast tissue.

Rapamycin effects on mTOR signaling in benign, premalignant and malignant human breast epithelial cells.

Rapamycin, an inhibitor of mTOR, is in clinical trials for treatment of cancer. Rapamycin resistance has been reported in human breast epithelial tumor cells. Rapamycin effects on mTOR signaling and resistance were examined using benign, premalignant and tumor human breast epithelial cells. Rapamycin inhibition of cell proliferation, the cell cycle and mTOR signaling, including p70S6 and S6RP phosphorylation, was most effective in benign (MCF10A) and premalignant (MCF10AT; MCF10ATG3B) human breast epithelial cells, relative to MCF10CA1a tumor cells. Rapamycin resistance was reflected by reduced inhibition of p70S6K and S6RP phosphorylation in MCF10CA1a tumor cells, with RS6P showing the least response to rapamycin in the tumor cells. Rapamycin differentially inhibited STAT3 phosphorylation in this cell lineage. These data suggest that inhibition of mTOR signaling and STAT3 phosphorylation in benign and premalignant cells may be effective in the treatment of proliferative breast disease (PBD) and in the prevention of tumorigenesis and tumor recurrence.

The role of intracellular signaling in insulin-mediated regulation of drug metabolizing enzyme gene and protein expression.

Endogenous factors, including hormones, growth factors and cytokines, play an important role in the regulation of hepatic drug metabolizing enzyme expression in both physiological and pathophysiological conditions. Diabetes, fasting, obesity, protein-calorie malnutrition and long-term alcohol consumption produce changes in hepatic drug metabolizing enzyme gene and protein expression. This difference in expression alters the metabolism of xenobiotics, including procarcinogens, carcinogens, toxicants and therapeutic agents, potentially impacting the efficacy and safety of therapeutic agents, and/or resulting in drug-drug interactions. Although the mechanisms by which xenobiotics regulate drug metabolizing enzymes have been studied intensively, less is known regarding the cellular signaling pathways and components which regulate drug metabolizing enzyme gene and protein expression in response to hormones and cytokines. Recent findings, however, have revealed that several cellular signaling pathways are involved in hormone- and growth factor-mediated regulation of drug metabolizing enzymes. Our laboratory has reported that insulin and growth factors regulate drug metabolizing enzyme gene and protein expression, including cytochromes P450 (CYP), glutathione S-transferases (GST) and microsomal epoxide hydrolase (mEH), through receptors which are members of the large receptor tyrosine kinase (RTK) family, and by downstream effectors such as phosphatidylinositol 3-kinase, mitogen activated protein kinase (MAPK), Akt/protein kinase B (PKB), mammalian target of rapamycin (mTOR), and the p70 ribosomal protein S6 kinase (p70S6 kinase). Here, we review current knowledge of the signaling pathways implicated in regulation of drug metabolizing enzyme gene and protein expression in response to insulin and growth factors, with the goal of increasing our understanding of how disease affects these signaling pathways, components, and ultimately gene expression and translational control.

Secretome analysis of microarray data reveals extracellular events associated with proliferative potential in a cell line model of breast disease.

It is widely believed that breast cancer develops in a multistep process with premalignant lesions preceding invasive carcinoma. The characterization of molecular events associated with premalignant progression would improve our understanding of carcinogenesis and greatly benefit the development of early detection methods and chemoprevention strategies. However, the molecular biology of precancerous breast disease is poorly understood. To better characterize extracellular events associated with disease progression, such as cell-cell and cell-extracellular matrix (ECM) signaling, we analyzed gene expression profiles for the set of genes coding for secreted proteins (the secretome) in a cell line model of human proliferative breast disease (PBD). PBD describes a series of preneoplastic changes in the inner lining of milk glands associated with a dramatic increase in the risk of breast cancer. We used a series of cell lines with increasing proliferative propensity, and cell cultures were grown on matrigel to emulate in vivo growth and ECM interactions. Microarray analysis identified two clusters of secretome genes with expression profiles correlating to PBD progression. Some of the identified genes have previously been associated with breast malignancies, and our results suggest that changes in expression for these genes begin in the premalignant stage, offering potential use for early detection and as chemotherapeutic targets. RT-PCR validation demonstrates the reliability of the microarray results.

Role of mechanical and redox stress in activation of mitogen-activated protein kinases in primary cultured rat hepatocytes.

Mechanical stress is known to activate signaling cascades, including mitogen-activated protein kinase (MAPK) pathways. Although mechanical stress has been implicated in hepatic cirrhosis and liver regeneration following hepatectomy, the signaling pathway(s) that may be activated in hepatocytes in response to mechanical stress have not been determined. Using primary cultured rat hepatocytes to examine cellular signaling in response to mechanical stress associated with medium change, we observed that the phosphorylation status of extracellular signal-regulated kinase 1/2 (ERK1/2), Jun N-terminal kinase and p38 MAPK, but not Akt, was altered. MAPK activation, especially ERK1/2, was rapidly increased within 5 min, followed by a subsequent decrease to below basal levels between 30 min and 1 h following medium change. MAPK/ERK kinase (MEK1/2) phosphorylation followed the same pattern. The phosphorylation of Raf-1 in response to medium change was also consistent with Raf-1 serving as an upstream regulator of MEK1/2-ERK1/2 signaling. Phosphorylation of ERK1/2 was increased by mechanical stress alone, suggesting that mechanical stress may be primarily responsible for ERK1/2 activation in response to medium change. Medium change produced a marked decline in oxidized glutathione and malondialdehyde levels, and the antioxidant N-acetyl-L-cysteine decreased basal ERK1/2 phosphorylation, suggesting a role for oxidative stress in maintaining basal ERK1/2 phosphorylation in cultured hepatocytes. These data suggest that medium change results in immediate activation of the MAPK signaling pathway due to mechanical stress, followed by a subsequent inactivation of MAPK signaling due to a reduction in oxidative stress levels. These processes may be associated with alteration of hepatic hemodynamic circulation observed in hepatic diseases and in liver transplantation.

Induction of microsomal epoxide hydrolase by sulfur amino acid deprivation via the pathway of C-Jun N-terminal kinase and its extracellular exposure during cell death.

Microsomal epoxide hydrolase (mEH), an epoxide detoxifying enzyme and putative cell surface autoantigen, is inducible by xenobiotics and by certain pathophysiological conditions (e.g., tumorigenesis and protein-calorie malnutrition). The present study was designed to determine mEH expression in H4IIE cells during cell death initiated by sulfur amino acid deprivation (SAAD) and to identify the signaling pathway for the enzyme induction. SAAD induced cell death at 48-72 h with translocation of Bax to mitochondria and increased mitochondrial permeability with cytochrome c release, both of which were prevented by SB203580 or by dominant-negative JNK1 [JNK1(-)] stable transfection. Caspase-3 activity was only marginally increased by SAAD. Neither genomic DNA fragmentation nor poly(ADP-ribose) polymerase cleavage was observed during SAAD-induced cell death. Thus, SAAD induced cell death independent of caspase activation. This was supported by the observation that benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a general caspase inhibitor, did not prevent cell death. The levels of mEH mRNA and protein were notably increased in cells under SAAD for 48-72 h. The induction of mEH occurred in parallel with cell death. Whereas SAAD-induced cell death resulted from both JNK1 and p38 kinase activation, mEH induction was decreased only by JNK1(-) transfection. Immunocytochemistry revealed that mEH protein was intensely stained in dying cells, cellular fragments and cell debris. Furthermore, the number of cells positive for surface mEH substantially increased by SAAD, as evidenced by flow cytometry analysis. These results demonstrated that SAAD induced nonapoptotic cell death with Bax translocation to mitochondria and mitochondrial cytochrome c release, but not through caspase-3 activation, and that mEH was induced by SAAD via the pathway of JNK1, but not ERK1/2 or p38 kinase, in parallel with cell death.

Gene-specific dye bias in microarray reference designs.

The most widely used microarray experiment design includes the use of a reference standard. Comparisons of gene expression between samples are facilitated because each sample is directly measured against the reference standard, using two fluorescent dyes. Numerous reports indicate that some genes incorporate the two commonly used dyes with different efficiencies, contributing to inaccurate data. However, it is widely assumed that these effects will not corrupt results if the reference standard is labeled with the same dye on each microarray. We demonstrate that this assumption is not reliable and that dye orientation can significantly influence measured changes in gene expression.

Oxidative DNA damage and repair in a cell lineage model of human proliferative breast disease (PBD).

Oxidative damage to DNA is thought to play a significant role in mutagenesis, aging, and cancer. Sensitivity to oxidative DNA damage and DNA repair efficiency were examined using a series of human breast epithelial cell lines-MCF-10A, MCF-10AT, and MCF-10ATG3B-with progressively elevated Ras protein. Breast epithelial cells were treated with H2O2, in the absence and presence of the DNA-repair inhibitors hydroxyurea (HU) and cytosine arabinoside (Ara-C). DNA strand breaks were assessed by the mean olive tail moment (microm) using the alkaline single-cell gel electrophoresis (Comet) assay. In untreated cells, the mean olive tail moment values were 4.3 +/- 0.7, 8.3 +/- 1.1, and 7.1 +/- 0.6 microm in the MCF-10A, MCF-10AT, and MCF-10ATG3B cells, respectively. Five min H2O2 treatment produced concentration-dependent DNA damage, with the MCF-10A cells most susceptible and the tumorigenic MCF-10ATG3B cells the least susceptible. Treatment with 100 microM H2O2 resulted in approximately 17-, 6-, and 4.5-fold increases in mean olive tail moment values in the MCF-10A, MCF-10AT, and MCF-10ATG3B cells, respectively, compared to untreated cells. The HCC1937 tumor cell line responded in a manner comparable to the MCF-10ATG3B cells treated with H2O2, HU/Ara-C pre-treatment resulted in a approximately 1.5-fold increase in olive tail moment values in all three cell lines. Protein levels of antioxidant enzymes, including catalase, copper/zinc superoxide dismutase (Cu/Zn SOD), and manganese SOD (MnSOD) were determined in order to examine a potential mechanism for increased resistance to H2O2-mediated DNA damage. Levels of these enzymes increased progressively, with highest expression in MCF-10ATG3B cells. Increased cellular resistance also coincided with marked decreases in p53 protein levels. These results demonstrate that, in this cell lineage, sensitivity to oxidative DNA damage by H2O2 decreases with tumorigenicity (i.e., MCF-10A vs. MCF-10ATG3B), and show that DNA repair, altered Ras, and p53 expression, or compensatory mechanisms involving elevated antioxidant enzymes are involved in mediating these effects.

Evaluation of the association between persistent organic pollutants (POPs) and diabetes in epidemiological studies: a national toxicology program workshop review.

BACKGROUND: Diabetes is a major threat to public health in the United States and worldwide. Understanding the role of environmental chemicals in the development or progression of diabetes is an emerging issue in environmental health. OBJECTIVE: We assessed the epidemiologic literature for evidence of associations between persistent organic pollutants (POPs) and type 2 diabetes. METHODS: Using a PubMed search and reference lists from relevant studies or review articles, we identified 72 epidemiological studies that investigated associations of persistent organic pollutants (POPs) with diabetes. We evaluated these studies for consistency, strengths and weaknesses of study design (including power and statistical methods), clinical diagnosis, exposure assessment, study population characteristics, and identification of data gaps and areas for future research. CONCLUSIONS: Heterogeneity of the studies precluded conducting a meta-analysis, but the overall evidence is sufficient for a positive association of some organochlorine POPs with type 2 diabetes. Collectively, these data are not sufficient to establish causality. Initial data mining revealed that the strongest positive correlation of diabetes with POPs occurred with organochlorine compounds, such as trans-nonachlor, dichlorodiphenyldichloroethylene (DDE), polychlorinated biphenyls (PCBs), and dioxins and dioxin-like chemicals. There is less indication of an association between other nonorganochlorine POPs, such as perfluoroalkyl acids and brominated compounds, and type 2 diabetes. Experimental data are needed to confirm the causality of these POPs, which will shed new light on the pathogenesis of diabetes. This new information should be considered by governmental bodies involved in the regulation of environmental contaminants.

Rapamycin sensitizes Akt inhibition in malignant human breast epithelial cells.

Akt and mTOR are therapeutic targets for the treatment of cancer. The effects of inhibiting mTOR, with rapamycin, and Akt, with A-443654, concurrently, on cell morphology, cell proliferation, the cell cycle, and apoptosis were examined using the benign MCF10A and malignant MCF10CA1a human breast epithelial cells. Rapamycin and A-443654 in combination produced the greatest morphological changes and inhibited cell proliferation by G2/M arrest. Rapamycin and A-443654 in combination induced apoptosis at earlier times and at lower A-443654 concentrations in MCF10CA1a tumor cells than in the benign MCF10A cells. Rapamycin and A-443654 increased p53 and p15(INK4B) protein levels, decreased anti-apoptotic Bcl-2 levels, and increased Bad levels in the MCF10CA1a tumor cells by approximately 5-fold. These results suggest that the combined inhibition of Akt and mTOR may have beneficial therapeutic and safety margin effects.

miRNAs: effectors of environmental influences on gene expression and disease.

Discovered less than a decade ago, micro-RNAs (miRNAs) have emerged as important regulators of gene expression in mammals. They consist of short nucleic acids, on average approximately 22 nucleotides in length. The miRNAs exert their effect by binding directly to target messenger RNAs (mRNAs) and inhibiting mRNA stability and translation. Each miRNA can bind to multiple targets and many miRNAs can bind to the same target mRNA, allowing for a complex pattern of regulation of gene expression. Once bound to their targets, miRNAs can suppress translation of the mRNA by either sequestration or degradation of the message. Thus, miRNAs function as powerful and sensitive posttranscriptional regulators of gene expression. This review will summarize what is known about miRNA biogenesis, expression, regulation, function, mode of action, and role in disease processes with an emphasis on miRNAs in mammals. We discuss some of the methodology employed in miRNA research and the potential of miRNAs as therapeutic targets. The role of miRNAs in signal transduction and cellular stress is reviewed. Lastly, we identify new exciting avenues of research on the role of miRNAs in toxicogenomics and the possibility of epigenetic effects on gene expression.

Identification of the insulin signaling cascade in the regulation of alpha-class glutathione S-transferase expression in primary cultured rat hepatocytes.

We reported previously that insulin elevated alpha-class glutathione S-transferase (GSTs) protein levels in primary cultured rat hepatocytes (Kim et al., 2003b). In contrast, glucagon down-regulated alpha- and pi-class GST expression, and mechanistic research implicated cAMP and protein kinase A in this process (Kim et al., 2003b). The present study examines the signaling pathways involved in the regulation of alpha-class GST in response to insulin in primary cultured rat hepatocytes. Protein levels of GSTA1/2 and GSTA3/5 and activity of GST toward 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD) were increased in an insulin concentration-dependent manner. Treatment of cells with the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] or rapamycin, an inhibitor of mammalian target of rapamycin and ribosomal p70 S6 kinase (p70S6K) phosphorylation, or with an adenovirus containing green fluorescent protein and a dominant-negative and kinase-dead Akt, effectively inhibited the insulin-mediated increase in alpha-class GST expression and GST activity toward NBD. In contrast, PD98059 (2'-amino-3'-methoxyflavone), an inhibitor of mitogen-activated protein kinase kinase, SP600125 (1,9-pyrazoloanthrone), an inhibitor of c-Jun N-terminal kinase, SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imadazole], an inhibitor of p38 mitogen-activated protein kinase, or bisindolylmaleimide, a broad spectrum inhibitor of protein kinase C, did not inhibit the insulin-mediated increase in alpha-class GST protein levels in hepatocytes. These results show that PI3K/Akt/p70S6K signaling is active in the insulin-mediated up-regulation of the antioxidant defense system and that low insulin levels, as encountered in diabetes, potentially increase the susceptibility of hepatocytes to xenobiotic-mediated and/or oxidative stress-mediated damage.

The mitogen-activated protein kinase kinase (mek) inhibitor PD98059 elevates primary cultured rat hepatocyte glutathione levels independent of inhibiting mek.

The antioxidant activity of flavonoids, directly through scavenging oxidizing species and indirectly through modulating drug-metabolizing enzyme activities, is associated with chemopreventive and chemotherapeutic effects. However, little published information is available concerning the effect of flavonoids on glutathione (GSH) homeostasis. We previously demonstrated that PD98059 (2'-amino-3'-methoxyflavone), a flavone derivative and selective mitogen-activated protein kinase kinase (MEK) 1 inhibitor, enhanced the insulin-mediated increase in GSH levels. To determine whether the PD98059-mediated increase in GSH levels was associated with MEK inhibition, primary cultured rat hepatocytes were treated with PD98059, the MEK inhibitor U0126, which is not a flavone derivative, or flavone. PD98059 increased GSH levels in a concentration-dependent manner in hepatocytes cultured in the presence or absence of insulin. In contrast, GSH levels were not affected by U0126 at concentrations sufficient to inhibit insulin-mediated extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Flavone, however, markedly increased GSH levels without inhibition of ERK1/2 phosphorylation. The concentration of GSH in the culture medium was also elevated by PD98059 or flavone, suggesting that the cellular GSH elevation could not be accounted for by the inhibition of GSH efflux into medium. Interestingly, PD98059 and flavone increased cellular cysteine levels, which may be responsible for the PD98059- and flavone-mediated elevation of GSH levels. These results provide evidence that PD98059 and flavone produce dramatic changes in GSH homeostasis in hepatocytes, through a mechanism(s) unrelated to MEK inhibition. Moreover, the current study implies that flavonoid-induced chemopreventive and chemotherapeutic effects may be mediated by regulation of redox state through the stimulation of GSH synthesis.

Acetoacetate induces CYP2E1 protein and suppresses CYP2E1 mRNA in primary cultured rat hepatocytes.

The ketone body acetoacetate (AA) in the absence of insulin or in the presence of diabetic insulin levels decreases CYP2E1 mRNA expression in a concentration- and time-dependent manner in primary cultured rat hepatocytes. AA activates p70 ribosomal S6 kinase (p70S6K) and protein kinase C (PKC) by approximately 2- to 2.5-fold, respectively, following 6-h treatment. The AA-mediated activation of p70S6K, but not PKC, was abolished by inhibition of PI 3-K with LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] or wortmannin, in agreement with p70S6K being downstream of phosphatidylinositol 3-kinase (PI 3-K). Inhibition of PI 3-K, mTOR with rapamycin, or PKC with bisindolylmaleimide ameliorated the AA-mediated down-regulation of CYP2E1 mRNA expression. Neither the mitogen-activated protein kinase kinase inhibitor PD98059 (2'-amino-3'-methoxyflavone) nor the p38 mitogen-activated protein kinase inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole] ameliorated the AA-mediated suppression of CYP2E1 mRNA expression. Heterogeneous nuclear RNA analysis revealed that AA suppressed CYP2E1 gene transcription by approximately 50% and that inhibition of PI 3-K and PKC diminished this AA-mediated effect on transcription. CYP2E1 mRNA half-life slightly increased from approximately 24 h in untreated hepatocytes to approximately 32 h in AA-treated cells. Interestingly, AA increased CYP2E1 protein levels by approximately 2- and 2.5-fold at 24 and 48 h, respectively. DL-beta-hydroxybutyrate was without effect. Polysomal distribution studies revealed that AA increased the proportion of RNA associated with the actively translated polysomal fractions versus the 40S to 60S untranslated fractions by approximately 40%. CYP2E1 protein half-life increased from approximately 8 h in untreated hepatocytes to approximately 24 in AA-treated cells. These data show that AA decreases CYP2E1 mRNA expression through inhibition of gene transcription while simultaneously elevating CYP2E1 protein levels through increased translation and decreased protein degradation.

Proceedings of a workshop on DNA adducts: biological significance and applications to risk assessment Washington, DC, April 13-14, 2004.

In April 2004, the Health and Environmental Sciences Institute, a branch of the International Life Sciences Institute, with support from the National Institute of Environmental Health Sciences, organized a workshop to discuss the biological significance of DNA adducts. Workshop speakers and attendees included leading international experts from government, academia, and industry in the field of adduct detection and interpretation. The workshop initially examined the relationship between measured adduct levels in the context of exposure and dose. This was followed by a discussion on the complex response of cells to deal with genotoxic insult in complex, interconnected, and interdependent repair pathways. One of the major objectives of the workshop was to address the recurring question about the mechanistic and toxicological relevance of low-concentration measured adducts and the presentations in the session entitled "Can low levels of DNA adducts predict adverse outcomes?" served as catalysts for further discussions on this subject during the course of the workshop. Speakers representing the regulatory community and industry reviewed the value, current practices, and limitations of utilizing DNA adduct data in risk assessment and addressed a number of practical questions pertaining to these issues. While no consensus statement emerged on the biological significance of low levels of DNA adducts, the workshop concluded by identifying the need for more experimental data to address this important question. One of the recommendations stemming from this workshop was the need to develop an interim "decision-logic" or framework to guide the integration of DNA adduct data in the risk assessment process. HESI has recently formed a subcommittee consisting of experts in the field and other key stakeholders to address this recommendation as well as to identify specific research projects that could help advance the understanding of the biological significance of low levels of DNA adducts.

Insulin and glucagon regulation of glutathione S-transferase expression in primary cultured rat hepatocytes.

Diabetes is a major cause of morbidity and mortality, and complications resulting from diabetes have been attributed in part to increased oxidative stress. Glutathione S-transferases (GSTs) constitute a major protective mechanism against oxidative stress. Studies of the expression and activity of GSTs during diabetes are inconclusive, with both increased and decreased GST expression being reported in vivo. Insulin and glucagon effects on GST expression and the signaling pathway involved in the glucagon regulation of GST expression were examined in primary cultured rat hepatocytes. The addition of insulin resulted in the elevation of alpha-class GST protein levels, whereas alpha- and pi-class GST protein levels were markedly decreased in hepatocytes cultured with glucagon. In contrast, mu-class GST protein expression was unaffected by insulin or glucagon treatment. Insulin concentrations >/=1 nM resulted in increased GST activities and alpha-class GST protein levels, whereas glucagon concentrations >/=20 nM decreased alpha- and pi-class protein levels and activity. Treatment of cells with 8-bromo-cAMP or dibutyryl-cAMP also resulted in decreased alpha- and pi-class GST protein levels. Pretreatment with N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide (H89), a selective inhibitor of protein kinase A, before glucagon addition markedly attenuated the glucagon effect. This study demonstrates that insulin and glucagon regulate, in an opposing manner, the expression of alpha-class GSTs and that glucagon completely inhibits pi-class GST expression in vitro, suggesting that hepatic GST expression may be decreased during diabetes. Furthermore, the present study implicates cAMP and protein kinase A in mediating the inhibitory effect of glucagon on GST expression.

Insulin signaling in the transcriptional and posttranscriptional regulation of CYP2E1 expression.

Diabetes has been reported to increase the expression of cytochrome P450 (CYP) 2E1 messenger RNA (mRNA) and protein several-fold, and enhanced expression has been associated with elevated ketone bodies. Primary cultured rat hepatocytes were used to explore ketone body and insulin regulation of CYP2E1 expression. Hydroxybutyrate and acetoacetate (AC), alone or in combination, either failed to affect or decreased CYP2E1 mRNA levels by up to 90% relative to untreated hepatocytes. Insulin produced a concentration-dependent decrease in CYP2E1 mRNA levels, and insulin receptor immunoprecipitation showed a correspondence between receptor phosphorylation and the decrease in CYP2E1 mRNA levels at physiologic levels of insulin. Phosphatase inhibitors decreased CYP2E1 mRNA levels by greater than 95%. The phosphatidylinositol 3-kinase (PI3-kinase) inhibitors wortmannin or LY294002 and rapamycin, an inhibitor of p70 S6 kinase phosphorylation, ameliorated the insulin-mediated decrease in CYP2E1 mRNA levels. Geldanamycin, which inhibits Src kinase, also abrogated the insulin-mediated decrease in CYP2E1 mRNA levels. In contrast, the protein kinase C (PKC) inhibitor bisindolylmaleimide, the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059, and the p38 mitogen-activated protein (MAP) kinase inhibitor SB202190 did not affect the insulin-mediated decrease in CYP2E1. CYP2E1 mRNA half-life decreased from approximately 48 hours in the absence of insulin to approximately 15 hours at 10 nmol/L insulin, and this decrease was prevented by wortmannin. The half-life of CYP2B mRNA was increased by insulin, whereas that of CYP3A was unaffected. Analysis of CYP2E1 gene transcription using heterogeneous nuclear RNA (hnRNA) showed that insulin suppressed CYP2E1 transcription. In conclusion, these data show involvement of transcriptional and posttranscriptional mechanisms in the insulin-mediated regulation of CYP2E1 and implicate PI3-kinase, p70 S6 kinase, and Src kinase in mediating these effects.

Acetoacetate activation of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase in primary cultured rat hepatocytes: role of oxidative stress.

Diabetes is characterized by elevated levels of ketone bodies acetoacetate (AA) and 3-hydroxybutyrate (3HB). High levels of ketone bodies have been implicated in generation of cellular oxidative stress. Ketone body activation of cellular signaling pathways associated with oxidative stress, however, has not been established. Thus, ketone body effects on kinase activation in primary cultured rat hepatocytes have been examined. Treatment with AA increased the phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2) and p38 mitogen-activated protein kinase (MAPK), maximally by approximately 2.5- and 4-fold, respectively. AA failed to activate c-Jun NH(2)-terminal kinase. AA-mediated Erk1/2 and p38 MAPK activation was detectable at 3 h post-treatment with maximal activation occurring at 12 h. In contrast, 3HB failed to activate any of these kinases. Elevated phosphorylation of Raf and MKK3/6 also occurred in response to AA. Bisindolylmaleimide, a generalized protein kinase C (PKC) inhibitor, and B581, a Ras farnesylation inhibitor, inhibited AA-mediated activation of Erk1/2 and p38 MAPK, suggesting a role for PKC and Ras in mediating such activation. Interestingly, the tyrosine kinase inhibitor genistein prevented the AA-mediated phosphorylation of Erk1/2, but not p38 MAPK. AA treatment resulted in the generation of reactive oxygen species (ROS) and the depletion of cellular glutathione levels, which was ameliorated by the antioxidants N-Acetyl-l-cysteine (NAC) and Trolox (6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid). NAC and Trolox also ameliorated AA-mediated Erk1/2 and p38 MAPK activation, suggesting that this activation is associated with ROS and oxidative stress.