Alkyladenine DNA glycosylase deficiency uncouples alkylation-induced strand break generation from PARP-1 activation and glycolysis inhibition.

DNA alkylation damage is repaired by base excision repair (BER) initiated by alkyladenine DNA glycosylase (AAG). Despite its role in DNA repair, AAG-initiated BER promotes cytotoxicity in a process dependent on poly (ADP-ribose) polymerase-1 (PARP-1); a NAD-consuming enzyme activated by strand break intermediates of the AAG-initiated repair process. Importantly, PARP-1 activation has been previously linked to impaired glycolysis and mitochondrial dysfunction.

Synergistic and antagonistic interactions of binary mixtures of polycyclic aromatic hydrocarbons in the upregulation of CYP1 activity and mRNA levels in precision-cut rat liver slices.

The current studies investigate whether synergistic or antagonistic interactions in the upregulation of CYP1 activity occur in binary mixtures of polycyclic aromatic hydrocarbons (PAHs) involving benzo[a]pyrene and five other structurally diverse PAHs of varying carcinogenic activity. Precision-cut rat liver slices were incubated with benzo[a]pyrene alone or in combination with a range of concentrations of a second PAH, and ethoxyresorufin O-deethylase, CYP1A1 and CYP1B1 mRNA levels determined. Concurrent incubation of benzo[a]pyrene with either dibenzo[a,h]anthracene or fluoranthene in liver slices led to a synergistic interaction, at least at low concentrations, in that ethoxyresorufin O-deethylase activity was statistically higher than the added effects when the slices were incubated with the individual compounds.

Inhibition of prenyltransferase activity by statins in both liver and muscle cell lines is not causative of cytotoxicity.

As inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase, statins are an important first-line treatment for hypercholesterolemia. However, a recognized side-effect of statin therapy is myopathy, which in severe cases can present as potentially fatal rhabdomyolysis. This represents an important impediment to successful statin therapy, and despite decades of research the molecular mechanisms underlying this side-effect remain unclear.

The naturally occurring aliphatic isothiocyanates sulforaphane and erucin are weak agonists but potent non-competitive antagonists of the aryl hydrocarbon receptor.

As the Ah receptor target gene products play a critical role in chemical carcinogenesis, antagonists are considered as potential chemopreventive agents. It is demonstrated in this paper that the isothiocyanates R,S-sulforaphane and erucin are non-competitive antagonists of the aryl hydrocarbon (Ah) receptor. Both isothiocyanates were poor agonists for the receptor and elevated CYP1A1 mRNA levels only modestly when incubated with precision-cut rat liver slices.

Phenethyl isothiocyanate, a naturally occurring phytochemical, is an antagonist of the aryl hydrocarbon receptor.

Scope: The aryl hydrocarbon (Ah) receptor is a ligand-activated transcription factor that is activated by many carcinogens, and its target gene products play a major role in tumour development, so that antagonists of the Ah receptor represent potential chemopreventive agents.

Methods And Results: Experimental evidence is presented herein that phenethyl isothiocyanate (PEITC), a phytochemical present in cruciferous vegetables, is such an antagonist. PEITC was a very weak ligand to the Ah receptor, as assessed using the chemical-activated luciferase expression (CALUX) assay, and a poor inducer of CYP1A1 mRNA levels when incubated in precision-cut rat liver slices for 24 h.

The neuroprotective action of the mood stabilizing drugs lithium chloride and sodium valproate is mediated through the up-regulation of the homeodomain protein Six1.

The mood stabilizing agents lithium chloride (LiCl) and sodium valproate (VPA) have recently gained interest as potential neuroprotective therapeutics. However, exploitation of these therapeutic applications is hindered by both a lack of molecular understanding of the mode of action, and a number of sub-optimal properties, including a relatively small therapeutic window and variable patient response. Human neuroblastoma cells (SH-SY5Y) were exposed to 1 mM lithium chloride or 1 mM sodium valproate for 6 h or 72 h, and transcriptomes measured by Affymetrix U133A/B microarray.

Up-regulation of the glutathione S-transferase system in human liver by polycyclic aromatic hydrocarbons; comparison with rat liver and lung.

The cytosolic glutathione S-transferases (GSTs) comprise a pivotal enzyme system protecting the cell from electrophilic compounds. It plays a major role in the detoxication of the primary and dihydrodiol epoxides of polycyclic aromatic hydrocarbons (PAHs), so that modulation of this enzyme system by PAHs will impact on their carcinogenic activity. The potential of six structurally diverse PAHs, namely benzo[a]pyrene (B[a]P), fluoranthene, benzo[b]fluoranthene (B[b]F), dibenzo[a,l]pyrene, dibenzo[a,h]anthracene (D[a,h]A) and 1-methhylphenanthrene, to modulate hepatic GST activity was investigated in human precision-cut slices and compared to rat slices, a species frequently used in long-term carcinogenicity studies; changes were monitored at the activity, using three different substrates, protein and mRNA levels.

Differential response of human and rat epoxide hydrolase to polycyclic aromatic hydrocarbon exposure: studies using precision-cut tissue slices.

The potential of polycyclic aromatic hydrocarbons (PAHs) to modulate microsomal epoxide hydrolase activity, determined using benzo[a]pyrene 5-oxide as substrate, in human liver, was evaluated and compared to rat liver. Precision-cut liver slices prepared from fresh human liver were incubated with six structurally diverse PAHs, at a range of concentrations, for 24h. Of the six PAHs studied, benzo[a]pyrene, dibenzo[a,h]anthracene and fluoranthene gave rise to a statistically significant increase in epoxide hydrolase activity, which was accompanied by a concomitant increase in epoxide hydrolase protein levels determined by immunoblotting.

Up-regulation of CYP1A/B in rat lung and liver, and human liver precision-cut slices by a series of polycyclic aromatic hydrocarbons; association with the Ah locus and importance of molecular size.

Exposure of precision-cut rat liver slices to six structurally diverse polycyclic aromatic hydrocarbons, namely benzo[a]pyrene, benzo[b]fluoranthene, dibenzo[a,h]anthracene, dibenzo[a,l]pyrene, fluoranthene and 1-methylphenanthrene, led to induction of ethoxyresorufin O-deethylase, CYP1A apoprotein and CYP1A1 mRNA levels, but to a markedly different extent. In liver slices, constitutive CYP1A1 mRNA levels were higher, as well as being markedly more inducible by PAHs, compared with CYP1B1, a similar profile to that observed in human liver slices following exposure to the PAHs. Increase in ethoxyresorufin O-deethylase and in CYP1A1 apoprotein levels was also observed when precision-cut rat lung slices were incubated with the same PAHs, the order of induction potency being similar to that observed in liver slices.

Evaluation of the precision-cut liver and lung slice systems for the study of induction of CYP1, epoxide hydrolase and glutathione S-transferase activities.

The principal objective was to ascertain whether precision-cut tissue slices can be used to evaluate the potential of chemicals to induce CYP1, epoxide hydrolase and glutathione S-transferase activities, all being important enzymes involved in the metabolism of polycyclic aromatic hydrocarbons. Precision-cut rat liver and lung slices were incubated with a range of benzo[a]pyrene concentrations for various time periods. A rise in the O-deethylation of ethoxyresorufin was seen in both liver and lung slices exposed to benzo[a]pyrene, which was accompanied by increased CYP1A apoprotein levels.

Steroidogenic gene expression in H295R cells and the human adrenal gland: adrenotoxic effects of lindane in vitro.

The focus on the refinement, reduction and replacement of animal use in toxicity testing requires the development of cell-based systems that mimic the effects of xenobiotics in human tissues. The human adrenocortical carcinoma cell line, H295R, has been proposed as a model for studies on adrenal steroidogenesis and its disruption. In this study, expression profiles for nine adrenal steroidogenic genes were characterized in H295R cells using real-time RT-PCR.

Transcriptomic and phylogenetic analysis of Kpna genes: a family of nuclear import factors modulated in xenobiotic-mediated liver growth.

Objectives: We have identified a member of the karyopherin (importin) alpha family of nuclear import factors as being modulated in rat liver following exposure to the hypolipidaemic and liver growth agent Wy-14,643. To examine the hypothetical role of this protein family as a checkpoint in receptor-mediated signalling, we characterized the rat karyopherin alpha (Kpna) gene family and present cDNA sequences and gene structures for all six rat Kpna genes. Further, we have assembled a comprehensive panel of Kpna coding regions from a range of metazoa, which we have subjected to phylogenetic analysis: This represents by far the most complete phylogenetic study of metazoan karyopherins, including several evolutionary intermediates not previously examined.

Strong polyadenylation and weak pausing combine to cause efficient termination of transcription in the human Ggamma-globin gene.

The human gamma-globin genes form part of a 5-kb tandem duplication within the beta-globin gene cluster on chromosome 11. Despite a high degree of identity between the two genes, we show that while the upstream Ggamma-globin gene terminates transcription efficiently, termination in the Agamma gene is inefficient. This is primarily due to the different strengths of the polyA signals of the two genes; Ggamma-globin has a functionally stronger polyA signal than the Agamma gene.

Gene expression changes in rat liver following exposure to liver growth agents: role of Kupffer cells in xenobiotic-mediated liver growth.

Many xenobiotics are known to cause liver enlargement and hepatocarcinogenesis in rats, although the molecular mechanisms that underlie this effect remain largely undefined. Human exposure to several of these compounds, including glucocorticoids and peroxisome proliferators may be significant, due to their use in both pharmaceutical and industrial processes. It is therefore important to elucidate the molecular mechanisms underlying this abnormal liver enlargement in rats, as this will enable more accurate extrapolation of the possible outcomes of human exposure.

Structure and transcriptional regulation of the Nat2 gene encoding for the drug-metabolizing enzyme arylamine N-acetyltransferase type 2 in mice.

Arylamine N-acetyltransferases (NATs) are polymorphic enzymes, well-known for their role in the metabolism of drugs and carcinogens. Mice have three NAT isoenzymes, of which NAT2 is postulated to be involved in endogenous, as well as xenobiotic, metabolism. To understand expression of the murine Nat2 gene, we have analysed its structure and transcriptional regulation.