Cytochrome P450-dependent Monooxygenase Research Articles

Species-specific activation of nuclear receptors correlates with the response of liver drug metabolizing enzymes to EMD 392949 in vitro

We previously reported on the species-specific effects on drug metabolizing enzymes (DME), in particular cytochrome P450-dependent monooxygenases (P450s), by the drug development candidate EMD 392949 (EMD) in vitro and in vivo. Induction of P450s occurs via activation of specific transcription factors such as the arylhydrocarbon receptor (AhR) and the nuclear xenobiotic receptors (NXRs). We analyzed whether the reported species-specific P450 induction by EMD could be related to a specific activation of the CYP1A regulator AhR and the CYP3A regulator pregnane X receptor (PXR) in human and rat cell lines. The human HepG2 and rat H4IIE cell lines exhibited inducibility of CYP1A and 3A and expressed functional AhR as well as PXR. CYP3A was induced by EMD in human HepG2 cells exceeding the level induced by rifampicin, but was not induced in rat H4IIE cells. Regulation of P450s was not related to expression levels of their respective transcription factor, but EMD treatment resulted in a significant reporter gene activation in xenobiotic response enhancer module (XREM)-transfected HepG2 but not H4IIE cells indicating activation of human but not rat PXR. In summary, we showed that the P450 inducing properties of EMD were perfectly reflected by its ability to activate AhR or PXR in a species-specific manner. These findings support the tight correlation of species-specific nuclear receptor activation with P450 induction and foster the use of nuclear receptor activation as a complementary screen to identify cytochrome P450 inducers.

Identification of a CYP3A form (CYP3A126) in fathead minnow (Pimephales promelas) and characterisation of putative CYP3A enzyme activity

Cytochrome P450-dependent monooxygenases (CYPs) are involved in the metabolic defence against xenobiotics. Human CYP3A enzymes metabolise about 50% of all pharmaceuticals in use today. Induction of CYPs and associated xenobiotic metabolism occurs also in fish and may serve as a useful tool for biomonitoring of environmental contamination. In this study we report on the cloning of a CYP3A family gene from fathead minnows (Pimephales promelas), which has been designated as CYP3A126 by the P450 nomenclature committee (GenBank no. EU332792). The cDNA was isolated, identified and characterised by extended inverse polymerase chain reaction (PCR), an alternative to the commonly used method of rapid amplification of cDNA ends. In a fathead minnow cell line we identified a full-length cDNA sequence (1,863 base pairs (bp)) consisting of a 1,536 bp open reading frame encoding a 512 amino acid protein. Genomic analysis of the identified CYP3A isoenzyme revealed a DNA sequence consisting of 13 exons and 12 introns. CYP3A126 is also expressed in fathead minnow liver as demonstrated by reverse transcription PCR. Exposure of fathead minnow (FHM) cells with the CYP3A inducer rifampicin leads to dose-dependent increase in putative CYP3A enzyme activity. In contrast, inhibitory effects of diazepam treatment were observed on putative CYP3A enzyme activity and additionally on CYP3A126 mRNA expression. This indicates that CYP3A is active in FHM cells and that CYP3A126 is at least in part responsible for this CYP3A activity. Further investigations will show whether CYP3A126 is involved in the metabolism of environmental chemicals.

Expression profiles of phases 1 and 2 metabolizing enzymes in human skin and the reconstructed skin models Episkin™ and full thickness model from Episkin™

Background Episkin™ and full thickness model from Episkin™ (FTM) are human skin models obtained from in vitro growth of keratinocytes into the five typical layers of the epidermis. FTM is a full thickness reconstructed skin model that also contains fibroblasts seeded in a collagen matrix. Objectives To assess whether enzymes involved in chemical detoxification are expressed in Episkin™ and FTM and how their levels compare with the human epidermis, dermis and total skin. Methods Quantification of the mRNA expression levels of phases 1 and 2 metabolizing enzymes in cultured Episkin™ and FTM and human epidermis, dermis and total skin using Realtime PCR. Results The data show that the expression profiles of 61 phases 1 and 2 metabolizing enzymes in Episkin™, FTM and epidermis are generally similar, with some exceptions. Cytochrome P450-dependent enzymes and flavin monooxygenases are expressed at low levels, while phase 2 metabolizing enzymes are expressed at much higher levels, especially, glutathione-S-transferase P1 (GSTP1) catechol-O-methyl transferase (COMT), steroid sulfotransferase (SULT2B1b), and N-acetyl transferase (NAT5). The present study also identifies the presence of many enzymes involved in cholesterol, arachidonic acid, leukotriene, prostaglandin, eicosatrienoic acids, and vitamin D3 metabolisms. Conclusion The present data strongly suggest that Episkin™ and FTM represent reliable and valuable in vitro human skin models for studying the function of phases 1 and 2 metabolizing enzymes in xenobiotic metabolisms. They could be used to replace invasive methods or laboratory animals for skin experiments.

Formation of UV-honey guides in Rudbeckia hirta

The UV-honey guides of Rudbeckia hirta were investigated by UV-photography, reflectance spectroscopy, LC–MS analysis and studies of the enzymes involved in the formation of the UV-absorbing flavonols present in the petals. It was shown for the first time that the typical bull’s eye pattern is already established at the early stages of flower anthesis on the front side of the petal surface, but is hidden to pollinators until the buds are open and the petals are unfolded. The rear side of the petals remains UV-reflecting during the whole flower anthesis. Studies on the local distribution of 19 flavonols across the petals confirmed that the majority are concentrated in the basal part of the ray flower. However, in contrast to the earlier studies, eupatolitin 3- O-glucoside (6,7-dimethoxyquercetin 3- O-glucoside) was present in both the basal and apical parts of the petals, whereas eupatolin (6,7-dimethoxyquercetin 3- O-rhamnoside) was exclusively found in the apical parts. The enzymes involved in the formation of the flavonols in R. hirta were demonstrated for the first time. These include a rare flavonol 6-hydroxylase, which was identified as cytochrome P450-dependent monooxygenase and did not accept any methylated flavonol as substrate. All enzymes were present in the basal and apical parts of the petals, although some of them clearly showed higher activities in the basal part. This indicates that the local accumulation of flavonols in R. hirta is not achieved by a locally restricted presence of the enzymes involved in flavonol formation.

The role of oxidative stress in alcoholic liver injury

Oxidative stress plays an important role in pathogenesis of alcoholic liver injury. The main source of free oxygen species is cytochrome P450-dependent monooxygenase, which can be induced by ethanol. ROLE OF CYTOCHROME P4502E1 IN ETHANOL-INDUCED OXIDATIVE STRESS: Reactive oxygen species produced by this enzyme are more important in intracellular oxidative damage compared to species derived from activated phagocytes. Free radicals lead to lipid peroxidation, enzymatic inactivation and protein oxidation. ROLE OF MITOCHONDRIA IN ALCOHOL-INDUCED OXIDATIVE STRESS. Production of mitochondrial reactive oxygen species is increased, and glutathione content is decreased in chronically ethanol-fed animals. Oxidative stress in mitochondria leads to mitochondrial DNA damage and has a dual effect on apoptosis. ROLE OF KUPFFER CELLS IN ALCOHOL-INDUCED LIVER INJURY: Chronic ethanol consumption is associated with increased release of endotoxin from gut lumen into portal circulation. Endotoxin activates Kupffer cells, which then release proinflammatory cytokines and oxidants. ROLE OF NEUTROPHILS IN ALCOHOL-INDUCED LIVER INJURY: Alcoholic liver injury leads to the accumulation of neutrophils, which release reactive oxygen species and lysosomal enzymes and contribute to hepatocyte damage and necrosis. ROLE OF NITRIC OXIDE IN ALCOHOL-INDUCED OXIDATIVE STRESS: High amounts of nitric oxide contribute to the oxidative damage, mainly by generating peroxynitrites. ROLE OF ANTIOXIDANTS IN ETHANOL-INDUCED OXIDATIVE STRESS: Chronic ethanol consumption is associated with reduced liver glutathione and alpha-tocopherol level and with reduced superoxide dismutase, catalase and glutathione peroxidase activity. Oxidative stress in alcoholic liver disease is a consequence of increased production of oxidants and decreased antioxidant defense in the liver.

Antidiabetic and ameliorative potential of Ficus bengalensis bark extract in streptozotocin induced diabetic rats

The aim of the present study was to evaluate the antidiabetic and ameliorative potential of aqueous extract of Ficus bengalensis bark in streptozotocin induced diabetic rats. The effect of oral administration of aqueous extract of F. bengalensis bark on blood glucose, serum electrolytes, serum glycolytic enzymes, liver microsomal protein, hepatic cytochrome P-450 dependent monooxygenase enzymes and lipid peroxidation in liver and kidney of streptozotocin -induced diabetic rats was studied. Oral administration of Ficus bengalensis to fed, fasted and glucose loaded diabetic rats significantly [F > 0.05 (ANOVA) and P< 0.05 (DMRT)] decreased the blood glucose level at 5 hrs and restored the levels of serum electrolytes, glycolytic enzymes and hepatic cytochrome P-450 dependent enzyme systems and decreased the formation of liver and kidney lipid peroxides at the end of 12 weeks. Further, the aqueous extract of Ficus bengalensis at a dose of 500mg/kg/day exhibits significant antidiabetic and ameliorative activity as evidenced by histological studies in normal and Ficus bengalensis treated streptozotocin induced diabetic rats. On the basis of our findings, it could be used as an antidiabetic and ameliorative agent for better management of diabetes mellitus.

Evaluation of Toluene Degradation Pathways by Two-Dimensional Stable Isotope Fractionation

Toluene degradation by several pure and mixed microbial cultures was investigated bytwo-dimensional compound specific isotope analysis (2D-CSIA). For most of the cultures, the respective toluene degradation pathway and toluene attacking enzymatic step was known. The slope of the linear regression for hydrogen (delta delta(2)H) vs. carbon (delta delta(13)C) discrimination (lamda = delta delta(2)H/ delta delta(13)C approximately epsilonH(bulk)/epsilonC(bulk)) was determined in order to characterize aerobic and anaerobic toluene degradation pathways. The highest lamda value was estimated for the monohydroxylation of the methyl group by Pseudomonas putida (lamda = 53 +/- 5). The lowest value was observed for Rhodococcus opacus (lamda = 2 +/- 2) due to its insignificant hydrogen fractionation, which indicates that a ring dioxygenase was responsible for the initial attack of toluene. The fungus Cladosprium sphaerospermum containing a cytochrome P450-dependent methyl monooxygenase grouped within these extreme values (lamda = 16 +/- 6). Lamda values for organisms attacking toluene under anoxic conditions by benzylsuccinate synthase were significantly different and ranged from lamda = 4 +/- 3 (Blastochloris sulfoviridis) to 31 +/- 11 (strain TRM1). Values were in the same range for organisms using nitrate (lamda = 11-14) or sulfate (lamda = 28-31) as electron acceptor, indicating that it might be possible to distinguish toluene degradation under different electron acceptor conditions by 2D-CSIA.

Precision-cut liver slices from rats of different ages: basal cytochrome P450-dependent monooxygenase activities and inducibility

The biotransformation capacity - of the cytochrome P450 (CYP) system for example - is lower but inducibility is more pronounced in neonates than in adults. On the other hand, both enzyme activities and inducibility decline with senescence. Precision-cut rat liver slices are widely used as an in vitro tool for the examination of drug toxicity, xenobiotic metabolism or enzyme induction. The aim of the present study was to assess whether age-related changes in CYP activities and induction observed in vivo are also mirrored in vitro in liver slices. For this purpose, different CYP model reactions were measured in precision-cut liver slices from one-day-old, 40-day-old and one-year-old rats after in vitro exposure to various inducers. Similar to the in vivo situation, basal CYP activities were distinctly lower and inducibility was much more pronounced in liver slices from neonatal than in those from adult animals. Also, enzyme activities were mostly somewhat lower in liver slices from aged rats compared to those from 40-day-old rats. However, CYP inducibility was less pronounced than with younger animals too. Thus, precision-cut rat liver slices are a suitable in vitro tool for investigating age-related changes in CYP activities and induction as well as developmental differences in drug metabolism and toxicity.

Biosynthesis of justicidin B and diphyllin in cell cultures of Linum perenne L. Himmelszelt

The arylnaphthalene lignans justicidin B (JusB) and diphyllin (Diph) exhibit several pharmaceutically interesting properties as antimicrobial, anti-inflammatory and anticancer activity. Cell suspension cultures of Linum perenne L. Himmelszelt accumulate JusB as the main component together with Diph-glycosides. A hypothetical biosynthetic pathway for these compounds is suggested. A cDNA encoding a pinoresinol-lariciresinol reductase (PLR-Lp1) which catalyses two early steps in the lignan biosynthesis was cloned from a cell culture of L. corymbulosum. Recombinant PLR-Lp1 prefers 8R, 8'R-pinoresinol in the first reaction step, but 8S, 8'S-lariciresinol in the second. We could prove its involvement in arylnaphthalene lignan biosynthesis in hairy roots of L. corymbulosum by an RNAi approach. Justicidin B 7-hydroxylase (JusB7H) catalyzes the last step in the biosynthesis of Diph by introducing a hydroxyl group in position 7 of JusB. This enzyme was characterized from a microsomal fraction prepared from a Linum perenne H suspension culture for the first time. The hydroxylase activity was strongly inhibited by cytochrome C as well as other cytochrome P450 inhibitors like clotrimazole indicating the involvement of a cytochrome P450-dependent monooxygenase. Justicidin B was the only substrate accepted by JusB7H with an apparent K m of 3.9±1.3µM. NADPH is predominantly accepted as the electron donor, but NADH was a weak cosubstrate. A synergistic effect of NADPH and NADH was not observed. The apparent K m for NADPH is 102±10µM.

Parallels in lignin biosynthesis

A hallmark of vascular plants is the development of a complex water-conducting system, which is physically reinforced by the heterogeneous aromatic polymer lignin. Syringyl lignin, a major building block of lignin, is often thought to be uniquely characteristic of angiosperms; however, it was demonstrated over fifty years ago that that syringyl lignin is found in another group of plants, known as the lycophytes, the ancestors of which diverged from all the other vascular plant lineages 400 million years ago.1 To determine the biochemical basis for this common biosynthetic ability, we isolated and characterized cytochrome P450-dependent monooxygenases (P450s) from the lycophyte Selaginella moellendorffii and compared them to the enzyme that is required for syringyl lignin synthesis in angiosperms. Our results showed that one of these P450s encodes an enzyme that is functionally analogous to but phylogenetically independent from its angiosperm counterpart. Here, we discuss the evolution of lignin biosynthesis in vascular plants and the role of Selaginella moellendorffii in plant comparative biology and genomics.

Independent origins of syringyl lignin in vascular plants.

Lycophytes arose in the early Silurian ( approximately 400 Mya) and represent a major lineage of vascular plants that has evolved in parallel with the ferns, gymnosperms, and angiosperms. A hallmark of vascular plants is the presence of the phenolic lignin heteropolymer in xylem and other sclerified cell types. Although syringyl lignin is often considered to be restricted in angiosperms, it has been detected in lycophytes as well. Here we report the characterization of a cytochrome P450-dependent monooxygenase from the lycophyte Selaginella moellendorffii. Gene expression data, cross-species complementation experiments, and in vitro enzyme assays indicate that this P450 is a ferulic acid/coniferaldehyde/coniferyl alcohol 5-hydroxylase (F5H), and is capable of diverting guaiacyl-substituted intermediates into syringyl lignin biosynthesis. Phylogenetic analysis indicates that the Selaginella F5H represents a new family of plant P450s and suggests that it has evolved independently of angiosperm F5Hs.

RNAi-mediated suppression of p -coumaroyl-CoA 3′-hydroxylase in hybrid poplar impacts lignin deposition and soluble secondary metabolism

p-Coumaroyl-CoA 3'-hydroxylase (C3'H) is a cytochrome P450-dependent monooxygenase that catalyzes the 3'-hydroxylation of p-coumaroyl shikimate and p-coumaroyl quinate. We used RNA interference to generate transgenic hybrid poplar suppressed in C3'H expression and analyzed them with respect to transcript abundance, cell wall structure and chemical composition, and soluble metabolite levels. RT-PCR expression profiles confirmed the down-regulation of C3'H in a number of lines, which generally correlated very well with reduced total cell wall lignin content. The most strongly repressed line was chosen for further analysis and compared with the wild-type trees. In-depth characterization revealed that along with the significant decrease in total lignin content, a significant shift in lignin monomer composition was observed, favoring the generation of p-hydroxyphenyl units at the expense of guaiacyl units while the proportion of syringyl moieties remained constant. Suppression of C3'H also resulted in the accumulation of substantial pools of 1-O-p-coumaroyl-beta-d-glucoside and other phenylpropanoid glycosides, and p-coumaroyl shikimate, providing further insight into the role of C3'H in the lignin biosynthetic pathway. The data presented indicate that when down-regulated, C3'H becomes a rate-limiting step in lignin biosynthesis and further support the involvement of hydroxycinnamic acid shikimate esters in the lignin biosynthetic pathway.

Effects of Cu 2+ and Pb 2+ on different fish species: Liver cytochrome P450-dependent monooxygenase activities and FTIR spectra

The effects of Cu 2+-sulfate and Pb 2+-acetate on carp ( Cyprinus carpio L.), silver carp ( Hypopthalmichtys molitrix V.) and wels ( Silurus glanis L.) were studied. The liver microsomal Cyt P450 content, the EROD, ECOD and APND monooxygenase activities were measured. In vivo treatment with 1 mg L − 1 Cu 2+ significantly elevated the activities of these enzymes and Cyt P450 content in silver carp livers. The high-dose Cu 2+ treatment (10 mg L − 1 ) on silver carp caused two-fold higher induction in the P450 dependent monooxygenase isoensymes than in wels. Although the 2 mg kg − 1 treatment with Pb 2+ in carp elevated significantly the P450 content, the EROD isoenzyme activities were significantly decreased after 1 day, showing the destructive effect of metal ion on the enzyme system. In vitro, Cu 2+ and Pb 2+ decreased the Cyt P450 content in the carp liver microsomes and the absorption peak shifted to higher wavelength. Fourier Transform Infrared (FTIR) spectroscopy was used to detect the damaging effects of the heavy metals. According to the inhibitory potency to Cu 2+, the most sensitive isoenzyme was the EROD in wels, the least was the silver carp's isoenzyme. The investigated fish P450 isoenzymes showed, that the Cu 2+ was a stronger inhibitor than Pb 2+.

Stem Cell Therapy for Liver Disease: Parameters Governing the Success of Using Bone Marrow Mesenchymal Stem Cells

Liver transplantation is the primary treatment for various end-stage hepatic diseases but is hindered by the lack of donor organs and by complications associated with rejection and immunosuppression. There is increasing evidence to suggest the bone marrow is a transplantable source of hepatic progenitors. We previously reported that multipotent bone marrow-derived mesenchymal stem cells differentiate into functional hepatocyte-like cells with almost 100% induction frequency under defined conditions, suggesting the potential for clinical applications. The aim of this study was to critically analyze the various parameters governing the success of bone marrow-derived mesenchymal stem cell-based therapy for treatment of liver diseases. Lethal fulminant hepatic failure in nonobese diabetic severe combined immunodeficient mice was induced by carbon tetrachloride gavage. Mesenchymal stem cell-derived hepatocytes and mesenchymal stem cells were then intrasplenically or intravenously transplanted at different doses. Both mesenchymal stem cell-derived hepatocytes and mesenchymal stem cells, transplanted by either intrasplenic or intravenous route, engrafted recipient liver, differentiated into functional hepatocytes, and rescued liver failure. Intravenous transplantation was more effective in rescuing liver failure than intrasplenic transplantation. Moreover, mesenchymal stem cells were more resistant to reactive oxygen species in vitro, reduced oxidative stress in recipient mice, and accelerated repopulation of hepatocytes after liver damage, suggesting a possible role for paracrine effects. Bone marrow-derived mesenchymal stem cells can effectively rescue experimental liver failure and contribute to liver regeneration and offer a potentially alternative therapy to organ transplantation for treatment of liver diseases.

Resistance to pyrethroid insecticides in Culex pipiens pipiens ( Diptera: Culicidae) from Tunisia

Resistance toward 2 pyrethroïd insecticides (permethrin and deltamethrin), and an organochloric one (DDT) was analyzed in 18 samples of Culex pipiens pipiens common mosquitoes collected from different Tunisian areas between March 2002 and November 2005. Bioassays were performed over different larvae samples. The recorded mortalities, after 24 h exposure to increased doses of insecticides, were compared to those obtained on a susceptible reference strain (S-LAB). All samples were resistant to permethrin. However, a large variation in the tolerance to this insecticide was observed between samples. Resistance ratio levels at LC 50 (RR 50) for the analyzed samples were ranged from 1.5 to 9092 folds. For deltamethrin, 14 among 17 studied samples were resistant. RR 50, in the resistant samples, were ranged from 3 to 453 folds. Resistance to the two tested pyrethroids and DDT were correlated. The use of synergists showed that the cytochrome P450-dependent monooxygenases had a major contribution to the permethrin and deltamethrin resistance and that the esterases (and/or glutathione- S-transferases) had only a minor contribution. Results were discussed in relation to resistance mechanisms and mosquito's control.

In vivo effects of Urtica urens (dwarf nettle) on the expression of CYP1A in control and 3-methylcholanthrene-exposed rats

The in vivo effects of the intraperitoneal administration of an Urtica urens L. (dwarf nettle) seed extract were examined on the hepatic, pulmonary, and renal cytochrome P450-dependent monooxygenase activities of rats co-administered with 3-methylcholanthrene (MC). Urtica extract was administered by intraperitoneal injection to male Wistar rats at 200mgkg−1day−1 for 4 days from which were also co-administered with intraperitoneal injection of 50mg of MCkg−1 of body weight twice on days 1 and 3. MC treatment increased the 7-ethoxyresorufin O-deethylase (EROD) activity of the liver, lung, and kidney 54-, 21-, and 119-fold, respectively. Urtica treatment substantially reduced the 3MC induction of hepatic, lung, and renal EROD activity by 79, 42, and 50%, respectively. Similarly, compared with the control, MROD activities in liver and kidney were increased after MC administration, and these increases were significantly inhibited by Urtica. reverse transcriptase-polymerase chain reaction (RT-PCR) analysis clearly showed that the hepatic CYP1A1 and CYP1A2 mRNA levels substantially increased after treatment with MC, which was suppressed by Urtica supplementation. Western blotting studies also supported the alterations observed in the catalytic activities and mRNA levels. In conclusion, substantial reduction in CYP1A1 and CYP1A2 expression levels and related activities with Urtica are possibly associated with a potential chemoprotective ability of the Urtica due to the anticipated decrease in the activation of environmental chemical carcinogens through modulation of the CYP1A enzymes.

Unexpected DNA damage caused by polycyclic aromatic hydrocarbons under standard laboratory conditions

The genotoxicity of 15 polycyclic aromatic hydrocarbons was determined with the alkaline version of the comet assay employing V79 lung fibroblasts of the Chinese hamster as target cells. These cells lack the enzymes necessary to convert PAHs to DNA-binding metabolites. Surprisingly, 11 PAHs, i.e., benzo[ a]pyrene (BaP), benz[ a]anthracene, 7,12-dimethylbenz[ a]anthracene, 3-methylcholanthrene, fluoranthene, anthanthrene, 11 H-benzo[ b]fluorene, dibenz[ a,h]anthracene, pyrene, benzo[ ghi]perylene and benzo[ e]pyrene caused DNA strand breaks even without external metabolic activation, while naphthalene, anthracene, phenanthrene and naphthacene were inactive. When the comet assay was performed in the dark or when yellow fluorescent lamps were used for illumination the DNA-damaging effect of the 11 PAHs disappeared. White fluorescent lamps exhibit emission maxima at 334.1, 365.0, 404.7, and 435.8 nm representing spectral lines of mercury. In the case of yellow fluorescent lamps these emissions were absent. Obviously, under standard laboratory illumination many PAHs are photo-activated, resulting in DNA-damaging species. This feature of PAHs should be taken into account when these compounds are employed for the initiation of skin cancer. The genotoxicity of BaP that is metabolically activated in V79 cells stably expressing human cytochrome P450-dependent monooxygenase (CYP1A1) as well as human epoxide hydrolase (V79-hCYP1A1-mEH) could not be detected with the comet assay performed under yellow light. Likewise the DNA-damaging effect of r-7, t-8-dihydroxy- t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[ a]pyrene ( anti-BaPDE) observed with the comet assay was only weak. However, upon inhibition of nucleotide excision repair (NER), which is responsible for the removal of stable DNA adducts caused by anti-BaPDE, the tail moment rose 3.4-fold in the case of BaP and 12.9-fold in the case of anti-BaPDE. These results indicate that the genotoxicity of BaP and probably of other compounds producing stable DNA adducts are reliably detected with the comet assay only when NER is inhibited.

Justicidin B 7-hydroxylase, a cytochrome P450 monooxygenase from cell cultures of Linum perenne Himmelszelt involved in the biosynthesis of diphyllin

Cell suspension cultures of Linum perenne L. Himmelszelt accumulate justicidin B as the main component together with glycosides of 7-hydroxyjusticidin B (diphyllin). A hypothetical biosynthetic pathway for these compounds is suggested. Justicidin B 7-hydroxylase (JusB7H) catalyzes the last step in the biosynthesis of diphyllin by introducing a hydroxyl group in position 7 of justicidin B. This enzyme was characterized from a microsomal fraction prepared from a Linum perenne Himmelszelt suspension culture for the first time. The hydroxylase activity was strongly inhibited by cytochrome c as well as other cytochrome P450 inhibitors like clotrimazole indicating the involvement of a cytochrome P450-dependent monooxygenase. JusB7H has a pH optimum of 7.4 and a temperature optimum of 26 °C. Justicidin B was the only substrate accepted by JusB7H with an apparent K m of 3.9 ± 1.3 μM. NADPH is predominantly accepted as the electron donor, but NADH was a weak co-substrate. A synergistic effect of NADPH and NADH was not observed. The apparent K m for NADPH is 102 ± 10 μM.

Hepatic Transcriptional Networks Induced by Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) serves as a prototype for a range of environmental toxicants and as a pharmacologic probe to study signal transduction by the aryl hydrocarbon receptor (AHR). Despite a detailed understanding of how TCDD exposure leads to the transcriptional up-regulation of cytochrome P450-dependent monooxygenases, we know little about how compounds like TCDD lead to a variety of AHR-dependent toxic end points such as liver pathology, terata, thymic involution, and cancer. Using an acute exposure protocol and the toxic response of the mouse liver as a model system, we have begun a detailed microarray analysis to describe the transcriptional changes that occur after various TCDD doses and treatment times. Through correlation analysis of time- and dose-dependent toxicological end points, we are able to identify coordinately responsive transcriptional events that can be defined as primary transcriptional events and downstream events that may represent mechanistically linked sequelae or that have potential as biomarkers of toxicity.

Detoxification Enzyme Activities in the FrogRana ridibundaas a Tool for Evaluating the Pollution of a Freshwater Ecosystem of Northern Greece with High Concentrations of Zinc and Copper

The accumulation of two metals (copper and zinc) and the specific activities of two enzyme systems glutathione S-transferases (GSTs) and cytochrome P450-dependent monooxygenases (P450-MO) were measured in four tissues (liver, kidney, small and large intestine) of the frog Rana ridibunda, collected from a wetland near the town of Lagadas, Northern Greece. Very high concentrations of the two metals were found in all studied tissues. The specific activities of both enzyme systems showed a significant decrease in frogs collected from the Lagada area compared to the controls. In the liver, both activities were reduced by about 88–90%. GST activity decreased by about 73% and 87% in the kidneys and small intestine, respectively, whereas the reduction in P450-MO activity was somewhat lower (about 59% in the kidney and 40% in the small intestine). Total protein correlated negatively with GST in the liver and kidney. Negative correlation was also found between copper and GST activity in the kidney. It is concluded ...