Cytochrome P450-dependent Activities Research Articles

Cytochrome P450-dependent drug oxidation activities and their expression levels in liver microsomes of chimeric TK-NOG mice with humanized livers

Hepatic cytochrome P450 (P450)-dependent drug oxidation activity has not been completely characterized in chimeric TK-NOG mice with humanized livers (humanized liver mice). In this study, we examined several drug oxidation activities catalyzed by liver microsomes from humans, humanized liver mice, and TK-NOG mice using 9 P450 substrates. The catalytic activities of liver microsomes from humans and humanized liver mice showed relatively similar rates of oxidation of 7-ethoxyresorufin, coumarin, 7-pentoxyresorufin, flurbiprofen, S-mephenytoin, chlorzoxazone, and midazolam, whereas bufuralol 1′-hydroxylation and propafenone 4′-hydroxylation (rodent-specific propafenone oxidation activity) were higher in humanized liver mice than in humans. In addition, P450 protein expression levels in the humanized mouse liver were quantified using a liquid chromatography–tandem mass spectrometry-based protein quantification method. Quantification of P450 enzymes showed a 3-fold difference between human and humanized liver mouse livers, except for CYP2B6, which showed an approximately 6-fold difference. Overall, most P450-dependent drug oxidation activities were comparable between liver microsomes from human and humanized liver mice based on the similar expression levels of human P450 enzymes. However, some differences were observed between both species, including considerable differences in bufuralol 1′-hydroxylation and propafenone 4′-hydroxylation activities.

The Foraging Gene, a New Environmental Adaptation Player Involved in Xenobiotic Detoxification.

Foraging is vital for animals, especially for food. In Drosophila melanogaster, this behavior is controlled by the foraging gene (for) which encodes a cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG). In wild populations of Drosophila, rover individuals that exhibit long foraging trails and sitter individuals that exhibit short ones coexist and are characterized by high and low levels of PKG activity, respectively. We, therefore, postulated that rover flies are more exposed to environmental stresses, including xenobiotics contamination, than sitter flies. We then tested whether these flies differed in their ability to cope with xenobiotics by exposing them to insecticides from different chemical families. We performed toxicological tests and measured the activity and expression levels of different classes of detoxification enzymes. We have shown that a link exists between the for gene and certain cytochrome P450-dependent activities and that the expression of the insecticide-metabolizing cytochrome P450 Cyp6a2 is controlled by the for gene. An unsuspected regulatory pathway of P450s expression involving the for gene in Drosophila is revealed and we demonstrate its involvement in adaptation to chemicals in the environment. This work can serve as a basis for reconsidering adaptation to xenobiotics in light of the behavior of species, including humans.

Biochemical aspects of susceptibility to stressors in two small cyprinids Squalius laietanus and Barbus meridionalis from the NW Mediterranean

Specimens of two endemic cyprinids, Squalius laietanus (Catalan chub) and Barbus meridionalis (Mediterranean barbel), were sampled from a reference site in a small stream of the Ripoll River (NW Mediterranean) outside of their reproductive season. Biomarkers involved in xenobiotic-mediated responses were individually contrasted in fish of both species and 17 perfluoroalkyl substances (PFASs) analysed in muscle to reveal bioaccumulation trends. The parameters were in muscle: cholinesterases, metabolic lactate dehydrogenase (LDH) and citrate synthase (CS); and in liver: cytochrome P450 dependent activities (EROD and BFCOD), carboxylesterase (CE), glutathione S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPX) and catalase (CAT). All markers are considered adaptive defence mechanism to face stress. Sensitivity to a model pesticide: dichlorvos was also contrasted in vitro in muscular acetylcholinesterase (AChE) and hepatic CE to reveal species sensitivity to neurotoxic chemicals. Enzymatic activities related to protective mechanisms such as butyrylcholinesterase (BuChE), CE and CAT were higher in chub whereas the antioxidant defences GR and GPX were higher in barbel. Aerobic CS was also higher in barbel while anaerobic LDH was so in chub. EROD activity did not differ between the two species but BFCOD activity was higher in barbel. Levels of PFAS were higher in barbel likely due to its benthic habitat. The in vitro tests revealed higher sensitivity to dichlorvos of muscular AChE in chub (lower IC50) which was probably compensated by a higher catalytic efficiency of CE. All these former biochemical particularities are discussed in terms of fish ecological performance in front of anthropogenic stressors.

Antioxidant properties and hepatoprotective effect of the edible halophyte Crithmum maritimum L. against carbon tetrachloride-induced liver injury in rats

A number of studies have shown the importance of halophyte species as sources of natural antioxidants. Crithmum maritimum is considered an edible and medicinal plant, though in vivo studies of this plant still are necessary to elucidate its potential benefits in alleviating toxicity. The present work was undertaken to investigate the major components and the antioxidant profile of C. maritimum leaf hydro-methanolic extract (CME) and the protective effects of C. maritimum (CM) against CCl4-induced toxicity in rats. Using LC–ESI–MS analysis, 17 phenolic compounds were identified in plant extract, with chlorogenic acid being the major component. The levels of total phenolic acids, total flavonoids and flavonols were ca. 26 mg GAE/g DW, 15.6 mg CE/g DW and 12 mg QE/g DW, respectively. Accordingly, CME showed important in vitro antioxidant activities (DPPH and ORAC). Moreover, CM supplementation to CCl4-intoxicated rats partially restored the impaired hepatic markers (ALT and AST activities and creatinine level), and reduced the CCl4-induced oxidative stress as shown by lipid peroxidation, GSH and protein carbonyl levels. The drug metabolizing system was also evaluated in liver by the measurement of some cytochrome P450-dependent activities such as ECOD, EROD and pNPH and the antioxidant DT-diaphorase, CAT and heme oxygenase activities. Thus, CM treatment reduced the enzymatic perturbations induced by CCl4 and hepatic damages observed from histopathological examination. The obtained results highlight the potential interest of C. maritimum as a source of bioactive compounds with relevant hepatoprotective effects.

Effects of tebuconazole on cytochrome P450 enzymes, oxidative stress, and endocrine disruption in male rats.

The major objective of the present study was to determine the ability of a triazole fungicide tebuconazole to induce cytochrome P450-dependent monooxygenases, oxidative stress, and endocrine-disrupting activity using male rats treated with tebuconazole at 10, 25, and 50 mg/kg p.o. once daily for 28 days. In liver, tebuconazole dose-dependently increased microsomal contents of cytochrome P450 and cytochrome b5 and the activities of NADPH-cytochrome P450 reductase, 7-ethoxyresorufin O-deethylase, methoxyresorufin O-demethylase, pentoxyresorufin O-dealkylase, 7-ethoxycoumarin O-deethylase, aniline hydroxylase, and erythromycin N-demethylase. In kidney, tebuconazole increased 7-ethoxycoumarin O-deethylase activity without affecting other monooxygenase activities. In marked contrast to liver and kidney, tebuconazole decreased testicular 7-ethoxyresorufin O-deethylase, methoxyresorufin O-demethylase, 7-ethoxycoumarin O-deethylase, aniline hydroxylase, and erythromycin N-demethylase activities. The results of immunoblot analysis of liver microsomes of controls and tebuconazole-treated rats revealed that tebuconazole induced CYP1A1/2, CYP2B1/2, CYP2E1, and CYP3A proteins in liver. Additions of tebuconazole to liver microsomes inhibited microsomal 7-ethoxycoumarin O-deethylase activity in vitro (IC50 = 1.50-1.69 µM). Treatment of rats with tebuconazole decreased glutathione content and increased glutathione S-transferase, superoxide dismutase, catalase, and glutathione peroxidase activities in liver; increased superoxide dismutase activities in kidney and testis; but decreased glutathione S-transferase activity in testis. Treatments with tebuconazole decreased serum testosterone concentration and cauda epididymal sperm count. The present study demonstrates that tebuconazole induces a multiplicity of CYPs and oxidative stress in liver; inhibits testicular P450 and glutathione S-transferase activities; and produces anti-androgenic effects in male rats.

Cytochrome P450-dependent drug oxidation activities in commercially available hepatocytes derived from human induced pluripotent stem cells cultured for 3 weeks.

Hepatocyte-like cells differentiated from human induced pluripotent stem (iPS) cells are of great interest for applications in pharmacological research. For drug metabolism testing, commercially available hepatocytes derived from human iPS cells are generally recommended to be used 1 week after seeding on plates. In this study, however, after 3-4 weeks of culture according to the manufacturer's instructions, human cytochrome P450 (P450) 2C9- and 2C19-dependent diclofenac 4'-hydroxylation and omeprazole 5-hydroxylation activities of the iPS-derived hepatocytes had significantly increased above the activities at 1 week and had reached levels similar to those in HepaRG cells, a human hepatocyte-like cell line. This increase in activities was associated with increasing P450 2C9 and 2C19 mRNA levels. Human P450 3A4-dependent midazolam 1'/4-hydroxylation activities in the iPS-derived hepatocytes were also enhanced after 3 weeks of culture, but the levels were low compared with those of HepaRG cells. These results indicate that the induction of mRNA of typical P450s in human iPS-derived hepatocyte-like cells occurred after 3 weeks of normal culture conditions. However, the induction levels varied considerably depending on the pregnane X receptor pathway and/or the P450 isoform. Our findings that the hepatic functions of human iPS-derived hepatocytes were enhanced by 3 weeks of simple culture could facilitate the use of these cells for drug metabolism and toxicity testing.

Induction of cytochrome P450-dependent mixed function oxidase activities and peroxisome proliferation by chloramine-T in male rat liver

Chloramine-T is an antimicrobial agent recognized for its disinfectant properties widely used in food industry. As an N-chloro-compound, chloramine-T contains electrophilic chlorine and in water it hydrolyses to hypochlorite. Chlorine as hypochlorous acid or hypochlorite is a very reactive chemical which can function as both an oxidant and halogenating agent. Because chloramine-T could be involved in the metabolic activation of drugs, in the present study the effects of chloramine-T on the activities of some drug metabolizing enzymes in rat liver microsomes and peroxisome proliferation were determined in vivo. Rats were treated orally with chloramine-T at doses of 1.25, 2.50, 5 and 10 mg/kg body weight (bw)/day for 6 days. The activities of CYP2E1, CYP1A1/2 CYP2B1/2, CYP3A4 and CYP4A1/2 enzymes significantly increased after treatment with 2.50, 5 and 10 mg/kg bw/day, in a dose-dependent manner as compared to control. This effect was not observed after chloramine-T treatment at dose of 1.25 mg/kg bw/day. Our results suggest that chloramine-T may potentiate the toxicity of many xenobiotics via metabolic activation and/or accumulation of reactive metabolites.

Progenitor-derived hepatocyte-like (B-13/H) cells metabolise 1'-hydroxyestragole to a genotoxic species via a SULT2B1-dependent mechanism.

Rat B-13 progenitor cells are readily converted into functional hepatocyte-like B-13/H cells capable of phase I cytochrome P450-dependent activation of pro-carcinogens and induction of DNA damage. The aim of the present study was to investigate whether the cells are also capable of Phase II sulphotransferase (SULT)-dependent activation of a pro-carcinogen to an ultimate carcinogen. To this end we therefore examined the bioactivation of the model hepatic (hepato- and cholangio-) carcinogen estragole and its proximate SULT1A1-activated genotoxic metabolite 1'-hydroxyestragole. Exposing B-13 or B-13/H cells to estragole (at concentrations up to 1mM) resulted in the production of low levels of 1'-hydroxyestragole, but did not result in detectable DNA damage. Exposing B-13/H cells - but not B-13 cells - to 1'-hydroxyestragole resulted in a dose-dependent increase in DNA damage in comet assays, confirmed by detection of N(2)-(trans-isoestragol-3'-yl)-2'-deoxyguanosine adducts. Genotoxicity was inhibited by general SULT inhibitors, supporting a role for SULTS in the activation of 1-hydroxyestragole in B-13/H cells. However, B-13 and B-13/H cells did not express biologically significant levels of SULT1A1 as determined by qRT-PCR, Western blotting and its associated 7-hydroxycoumarin sulphation activity. B-13 and B-13/H cells expressed - relative to intact rat liver - high levels of SULT2B1 (primarily the b isoform) and SULT4A1 mRNAs and proteins. B-13 and B-13/H cells also expressed the 3'-phosphoadenosine 5'-phosphosulphate synthase 1 required for the generation of activated sulphate cofactor 3'-phosphoadenosine 5'-phosphosulphate. However, only B-13/H cells expressed functional SULT activities towards SULT2B1 substrates DHEA, pregnenolone and 4 methylumbelliferone. Since liver progenitor cells are bi-potential and also form cholangiocytes, we therefore hypothesised that B-13 cells express a cholangiocyte-like SULT profile. To test this hypothesis, the expression of SULTs was examined in liver by RT-PCR and immunohistochemistry. SULT2B1 - but not SULT1A1 - was determined to be expressed in both rat and human cholangiocytes. Since 1'-hydroxyestragole exposure readily produced DNA injury in B-13/H cells, these data suggest that cholangiocarcinomas generated in rats fed estragole may be dependent, in part, on SULT2B1 activation of the 1'-hydroxyestragole metabolite.

Endocrine activities and cellular stress responses in the marsh frog Pelophylax ridibundus exposed to cobalt, zinc and their organic nanocomplexes

Metal-containing materials are extensively used in industry, personal care products and medicine, and their release in the environment causes concern for the potential impacts on aquatic organisms. We assessed endocrine disrupting potential of N-vinyl-2-pyrrolidone-based nanoparticles (Me-PSs) containing cobalt (Co2+) or zinc (Zn2+), using the marsh frog Pelophylax ridibundus as a model. Adult males were exposed for 14 days to waterborne Co2+ (50μg/L), Zn2+ (100μg/L) or corresponding concentrations of Co-PS, Zn-PS, or parental polymeric compound (PS). The indices of thyroid activity, vitellogenesis, cytochrome P450-dependent monooxygenases activity (EROD) and cytotoxicity markers were evaluated. Exposure to Co2+ led to the elevation of serum thyrotropin (TSH) and hepatic deiodinase activities accompanied by the up-regulation of EROD activity. In contrast, the action of the polymer-containing substances (Co-PS, Zn-PS and PS) as well as free Zn2+ caused a prominent decrease of EROD activity and a decrease in serum cortisol and TSH concentrations. Exposures to Zn2+, Zn-PS and PS upregulated vitellogenesis in males. All exposures except Co2+ caused neurotoxicity as indicated by the depletion of cholinesterase. These results demonstrate toxicity of Co- and Zn-containing Me-PSs and their parental compounds (Zn2+ and PS) in frogs and indicate distinct mechanisms of Co2+ action. Broad disruption of the hormonal pathways and reduced capacity for organic xenobiotic detoxification may have deleterious impacts on amphibian populations from habitats exposed to metallorganic pollution.

Status of pyrethroid resistance and mechanisms in Brazilian populations of Tuta absoluta

The tomato leafminer, Tuta absoluta, is a major pest of tomato crops worldwide. This study surveyed the resistance of T. absoluta populations from four regions in Brazil to pyrethroid insecticides, the frequencies of L1014F, T929I and M918T Na channel mutations, and the role of detoxification metabolism in the resistance. Resistance ratios varied from 1- to 11-times among populations and insecticides, but control failure likelihood assays showed that all pyrethroids assessed exhibited no efficacy at all (and thus, 98–100% control failure likelihood) against all T. absoluta populations. The activity of glutathione S-transferase and cytochrome P450-mediated N-demethylation in biochemical assays was significantly correlated with the level of resistance to deltamethrin and permethrin suggesting that these enzymes may play a role in resistance. TaqMan assays were used to screen for the presence of knockdown resistance (kdr) mutations and revealed that the L1014F kdr mutation was fixed in all populations and associated with two super-kdr mutations, M918T and particularly T929I, at high frequency. Altogether, results suggest that control failures are because of mutations in the domain II of the sodium channel, as a prevailing mechanism of resistance to pyrethroids in populations of T. absoluta in Brazil. But, enhanced cytochrome P450-dependent monooxygenases and GST activities also play an important role in the resistance of some populations, which reinforce that pyrethroids must not be used overall to control T. absoluta.

Applying of hierarchical clustering to analysis of protein patterns in the human cancer-associated liver.

BackgroundThere are two ways that statistical methods can learn from biomedical data. One way is to learn classifiers to identify diseases and to predict outcomes using the training dataset with established diagnosis for each sample. When the training dataset is not available the task can be to mine for presence of meaningful groups (clusters) of samples and to explore underlying data structure (unsupervised learning).ResultsWe investigated the proteomic profiles of the cytosolic fraction of human liver samples using two-dimensional electrophoresis (2DE). Samples were resected upon surgical treatment of hepatic metastases in colorectal cancer. Unsupervised hierarchical clustering of 2DE gel images (n = 18) revealed a pair of clusters, containing 11 and 7 samples. Previously we used the same specimens to measure biochemical profiles based on cytochrome P450-dependent enzymatic activities and also found that samples were clearly divided into two well-separated groups by cluster analysis. It turned out that groups by enzyme activity almost perfectly match to the groups identified from proteomic data. Of the 271 reproducible spots on our 2DE gels, we selected 15 to distinguish the human liver cytosolic clusters. Using MALDI-TOF peptide mass fingerprinting, we identified 12 proteins for the selected spots, including known cancer-associated species.Conclusions/SignificanceOur results highlight the importance of hierarchical cluster analysis of proteomic data, and showed concordance between results of biochemical and proteomic approaches. Grouping of the human liver samples and/or patients into differing clusters may provide insights into possible molecular mechanism of drug metabolism and creates a rationale for personalized treatment.

Comparison of xenobiotic metabolizing enzyme activities in ex vivo human skin and reconstructed human skin models from SkinEthic

Skin function is not limited to a physical barrier. According to its total surface area, it is also considered as an extra-hepatic metabolizing organ. In vitro engineered human skins have been developed to replace limited ex vivo normal human skin samples (NHS). Thus, assessing and comparing skin models from SkinEthic [Episkin™, RHE™ and the full thickness model (FTM)] with NHS in terms of metabolic capability are essential. The apparent activities of main cutaneous isoforms of cytochrome P450-dependent monooxygenases (CYP1A1/1B1, 2B6/2C18/2E1, 3A5/3A7), esterase, glutathione-S-[(GST), A, M, P, T], N-acetyl-(NAT1), uridinyl-diphosphate glucuronyl-(UDPGT 1A family) and sulfo-(SULT1A1) transferases were determined using probe substrates. Mean activities indicative of CYP1A1/1B1 (expressed as pmol/mg protein/6 h) in RHE™ (2.8) and FTM (2.6) were very similar to NHS (3.0) while Episkin™ showed a higher activity (9.1). Activities of CYP3A5/3A7 in FTM (3.3) and Episkin™ (3.6) were similar to NHS (3.8) while activity in RHE™ (13.3) was higher. CYP2B6/2C18/2E1 activity was below LOQ (0.5) in all skin models and NHS. Comparable intrinsic metabolic clearances were measured between NHS and skin models for esterase, UDPGT, GST and NAT1 activities. SULT1A1 activity toward probe substrates was not detected in skin models and observed at the limit of detection in NHS. Weak cytochrome P450-dependent monooxygenases, high esterase and transferase activities suggested that NHS and skin models exhibited limited functionalization and much greater detoxification (hydrolytic and conjugating) capacities. These results demonstrate that skin models are similar to NHS in terms of metabolic functionality toward xenobiotics investigated and useful tools to assess both the local efficiency and safety of cosmetics.

A Novel Mouse Model for Phenytoin-Induced Liver Injury: Involvement of Immune-Related Factors and P450-Mediated Metabolism

Drug-induced liver injury is an important issue for drug development and clinical drug therapy; however, in most cases, it is difficult to predict or prevent these reactions due to a lack of suitable animal models and the unknown mechanisms of action. Phenytoin (DPH) is an anticonvulsant drug that is widely used for the treatment of epilepsy. Some patients who are administered DPH will suffer symptoms of drug-induced liver injury characterized by hepatic necrosis. DPH-induced liver injury occurs in 1 in 1000 or 1 in 10 000 patients. Clinically, 75% of patients who develop liver injury develop a fever and 63% develop a rash. In this study, we established a mouse model for DPH-induced liver injury and analyzed the mechanisms for hepatotoxicity in the presence of immune-related or inflammation-related factors and metabolic activation. Female C57BL/6 mice were administered DPH for 5 days in combination with L-buthionine-S,R-sulfoximine. Then, the plasma alanine aminotransferase (ALT) levels were increased, hepatic lesions were observed during the histological evaluations, the hepatic glutathione levels were significantly reduced, and the oxidative stress marker levels were significantly increased. The inhibition of cytochrome P450-dependent oxidative metabolism significantly suppressed the elevated plasma ALT levels and depleted hepatic glutathione. Among the innate immune factors, the hepatic mRNA levels of NACHT, LRR, pyrin domain-containing protein 3, interleukin-1β, and damage-associated molecular patterns were significantly increased. Prostaglandin E₁ treatment ameliorated the hepatic injury caused by DPH. In conclusion, cytochrome P450-dependent metabolic activation followed by the stimulation of the innate immune responses is involved in DPH-induced liver injury.

Abstract 3596: Short-term effect of nose-only cigarette smoke exposure on redox homeostasis and mitochondrial metabolism in mice.

Abstract The components of cigarette smoke have been implicated in the development of cancer as well as in cardiovascular diseases. We have previously reported oxidative stress in rat tissues induced by tobacco-specific toxins nicotine and 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Recently, we have also shown increased oxidative stress and associated inflammatory responses in various tissues after exposure to cigarette smoke. In this study, we have further investigated the effects of nose-only cigarette smoke exposure on mitochondrial functions and glutathione-dependent redox metabolism in tissues of BALB/C mice. Liver, kidney, heart and lung tissues were analyzed for oxidative stress, glutathione (GSH) and cytochrome P450 dependent enzyme activities and mitochondrial functions after exposure to smoke generated by 9 cigarettes/day for 4 days. Control mice were exposed to air only. An increase in oxidative stress as observed by an increased production of reactive oxygen species (ROS) and altered GSH metabolism was apparent in all the tissues, but lung and heart appeared to be the main targets affected. Increased expression and activity of CYP450 1A1 and 1A2 were also observed in the tissues after exposure to cigarette smoke. Mitochondrial respiratory functions in the tissues, as observed by alterations in the activities of Complex I and IV enzymes, was also observed after exposure to cigarette smoke when compared with air only exposed mice. SDS-PAGE and Western blot results also indicate that alterations in the expression of enzyme proteins were in accordance with the changes in their catalytic functions. These results suggest that even short term exposure of cigarette smoke have adverse effects on mitochondrial functions and redox homeostasis in tissues which may progress to further complications associated with chronic smoking. (The study is supported by Terry Fox Cancer Research Funds and by the Research Committee Grants from College of Medicine and Health Sciences) Citation Format: Haider Raza, Annie John, Abderrahim Nemmar. Short-term effect of nose-only cigarette smoke exposure on redox homeostasis and mitochondrial metabolism in mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3596. doi:10.1158/1538-7445.AM2013-3596

Short-Term Effects of Nose-Only Cigarette Smoke Exposure on Glutathione Redox Homeostasis, Cytochrome P450 1A1/2 and Respiratory Enzyme Activities in Mice Tissues

Background/Aims: The components of cigarette smoke (CS) have been implicated in the development of cancer as well as in cardiopulmonary diseases. We have previously reported increased oxidative stress in rat tissues induced by tobacco-specific toxins nicotine and 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Recently, we have also shown increased oxidative stress and associated inflammatory responses in various tissues after exposure to cigarette smoke. Methods: In this study, we have further investigated the effects of nose-only cigarette smoke exposure on mitochondrial functions and glutathione-dependent redox metabolism in tissues of BALB/C mice. Liver, kidney, heart and lung tissues were analyzed for oxidative stress, glutathione (GSH) and cytochrome P450 dependent enzyme activities and mitochondrial functions after exposure to smoke generated by 9 cigarettes/day for 4 days. Control mice were exposed to air only. Results: An increase in oxidative stress as observed by increased production of reactive oxygen species (ROS) and altered GSH metabolism was apparent in all the tissues, but lung and heart appeared to be the main targets. Increased expression and activity of CYP450 1A1 and 1A2 were also observed in the tissues after exposure to cigarette smoke. Mitochondrial respiratory dysfunction in the tissues, as observed by alterations in the activities of Complex I and IV enzymes, was also observed after exposure to cigarette smoke. SDS-PAGE and Western blot results also indicate that alterations in the expression of enzyme proteins were in accordance with the changes in their catalytic functions. Conclusion: These results suggest that even short term exposure of cigarette smoke have adverse effects on mitochondrial functions and redox homeostasis in tissues which may progress to further complications associated with chronic smoking.

NCTC 2544 and IL-18 production: A tool for the identification of contact allergens

Progress in understanding the mechanisms of skin sensitization, provides us with the opportunity to develop in vitro tests as an alternative to in vivo sensitization testing.Keratinocytes play a key role in all phases of skin sensitization. We have recently identified interleukin-18 (IL-18) production in keratinocyte as a potentially useful endpoint for determination of contact sensitization potential of low molecular weight chemicals. The aim of the present article is to further exploit the performance of the NCTC 2544 assay.NCTC 2544 is a commercially available skin epithelial-like cell line originating from normal human skin, which posses a good expression of cytochrome P450-dependent enzymatic activities. Cells were exposed to contact allergens (2-bromo-2-bromomethyl glutaronitrile, cinnamaldehyde, citral, diethylmaleate, dinitrochlorobenzene, glyoxal, 2-mercaptobenzothiazole, nickel sulfate, 4-nitrobenzylbromide, oxazolone, penicillin G, resorcinol, tetramethylthiuram disulfide), to pre- pro-haptens (cinnamyl alcohol, eugenol, isoeugenol, p-phenylediamine), to respiratory allergens (ammonium hexachloroplatinate, diphenylmethane diisocyanate, glutaraldehyde, hexamethylenediisocyanate, maleic anhydride, trimellitic anhydride) and to irritants (benzaldehyde, cholorobenzene, diethylphtalate, hydrobenzoic acid, lactic acid, octanoic acid, phenol, salicylic acid, sodium lauryl sulphate, sulfamic acid). Cell associated IL-18 was evaluated 24 later by ELISA. At not-cytotoxic concentrations (cell viability higher of 80%, as assessed by MTT reduction assay), all contact sensitizers, including pre-pro-haptens, induced a dose-related increase in IL-18, whereas both irritants, with the exception of sulfamic acid, and respiratory allergens failed. A total of 33 chemicals were tested, with an overall accuracy of 97%.Overall, results obtained indicated that cell-associated IL-18 might provide an in vitro tool for identification and discrimination of contact vs. respiratory allergens and/or irritants.

An in vitro tool to assess cytochrome P450 drug biotransformation-dependent cytotoxicity in engineered HepG2 cells generated by using adenoviral vectors

Many adverse drug reactions leading to hepatotoxicity are caused by the cytochrome P450-dependent activation of non-toxic drugs or chemicals into reactive metabolites. To this end, adenoviruses were used as a tool to efficiently deliver specific CYP genes into cultured cells (i.e., human hepatoma cell line HepG2). Recombinant-defective adenoviral vectors encoding for genes CYP3A4 (Adv-CYP3A4), CYP2E1 (Adv-CYP2E1), CYP2A6 (Adv-CYP2A6) and CYP1A2 (Adv-CYP1A2) were used to confer specific CYP drug metabolic capabilities to HepG2 cells. Upgraded cells transiently expressed single specific cytochrome P450 enzymatic activities in terms of the number of the infecting virus particles used in their transduction. HepG2 cells transduced with adenoviruses and wild HepG2 cells cultured in 96 well-plates were incubated in the presence of model compounds, some of which can be metabolized to reactive metabolites. After compound exposure, cell viability was assessed by the commonly used MTT assay. The results confirm that the cell-based assay is a valuable tool in toxicology assessments and high-throughput screenings to detect cytotoxicity mediated by cytochrome P450 biotransformation in preclinical drug development. The assay also has a potential applicability in other industrial sectors such as the chemical industry.

Assessment of RTG-W1, RTL-W1, and PLHC-1 fish cell lines for genotoxicity testing of environmental pollutants by means of a Fpg-modified comet assay

In a context of growing awareness of aquatic pollution impacts, there is an increasing need to develop methods for hazard and risk assessment of pollutants. For this purpose, in vitro models such as fish cell lines warrant to be evaluated as possible alternative to in vivo fish testing, and new toxicity endpoints such as genotoxicity deserve to be considered. This study assesses the interest of the formamido pyrimidine glycosylase (Fpg)-modified comet assay applied to three fish cell lines (RTL-W1, RTG-W1, and PLHC-1) regarding the sensitivity of the system for measuring genotoxicity of various classes of pollutants. Cytochrome P450-dependent EROD activity has also been measured to evaluate the importance of the biotransformation capacity of the cell lines in genotoxicity assessment. For all cell lines and chemicals tested, a concentration dependent genotoxic effect was observed with a 10- to 1000-fold increased sensitivity when using the Fpg-protocol compared to the standard comet assay. Such a modified assay led in particular to improve the detection threshold of oxidative and alkylating DNA damages following exposure at environmentally relevant contaminant concentrations and could partly compensate for the lower sensitivity of cell lines versus whole organism testing often cited as a limit of in vitro testing.

Gender-related differences in cytochrome P450 in porcine liver--implication for activity, expression and inhibition by testicular steroids.

In pigs, the hepatic cytochrome P450 (CYP) 1A2, 2A and 2E1 activity is important in the regulation of skatole accumulation in adipose tissue. This study investigated gender-related differences in CYP1A2, 2A and 2E1 dependent activity, protein and mRNA expression. This study also investigated the gonadal steroid dependent inhibition of CYP activity in relation to gender and dietary composition. Microsomes were prepared from the liver of female and entire male pigs (Landrace × Yorkshire sire and Duroc boars) reared under similar conditions and slaughtered at an age of 164 days. A group of entire male pigs fed dried chicory root for 16 days prior to slaughter were included in the study. CYP activities were assessed by the use of probe substrates, whilst mRNA and protein expression were analysed by RT-PCR and Western blotting. Furthermore inhibition of CYP dependent activity by gonadal steroids was assessed in vitro. Microsomes from female pigs had greater CYP1A2 and 2A activity, as well as mRNA expression compared to entire male pigs. The antibodies used did not detected differences in protein expression. In vitro inhibition by 17β-oestradiol, oestrone, androstenone and 3β-OH androstenol of CYP2E1 activity in microsomes from entire male pigs as well as inhibition of CYP1A activity in chicory fed entire male pigs was observed. Apart from that no effect of steroids was shown. In conclusion, female pigs show greater CYP activity and mRNA expression. Including chicory in the diet for 16 days changed the gonadal steroid dependent inhibition of CYP activity in entire male pigs.

Protein Thiol Oxidation in Murine Airway Epithelial Cells in Response to Naphthalene or Diethyl Maleate

Naphthalene (NA) is a semivolatile aromatic hydrocarbon to which humans are exposed from a variety of sources. NA results in acute cytotoxicity to respiratory epithelium in rodents. Cytochrome P450-dependent metabolic activation to form reactive intermediates and loss of soluble cellular thiols (glutathione) are critical steps in NA toxicity, but the precise mechanisms by which this chemical results in cellular injury remain unclear. Protein thiols are likely targets of reactive NA metabolites. Loss of these, through adduction or thiol oxidation mechanisms, may be important underlying mechanisms for NA toxicity. To address the hypothesis that loss of thiols on specific cellular proteins is critical to NA-induced cytotoxicity, we compared reduced to oxidized thiol ratios in airway epithelial cell proteins isolated from lungs of mice treated with NA or the nontoxic glutathione depletor, diethyl maleate (DEM). At 300 mg/kg doses, NA administration resulted in a greater than 85% loss of glutathione levels in the airway epithelium, which is similar to the loss observed after DEM treatment. Using differential fluorescent maleimide labeling followed by 2DE separation of proteins, we identified more than 35 unique proteins that have treatment-specific differential sulfhydryl oxidation. At doses of NA and DEM that produce similar levels of glutathione depletion, Cy3/Cy5 labeling ratios were statistically different for 16 nonredundant proteins in airway epithelium. Proteins identified include a zinc finger protein, several aldehyde dehydrogenase variants, beta-actin, and several other structural proteins. These studies show distinct patterns of protein thiol alterations with the noncytotoxic DEM and the cytotoxic NA.