Establishment of a method to expose and measure pollution in excised porcine skin with electron paramagnetic resonance spectroscopy.

Health problems associated with the amount of air pollutants are increasing worldwide. Pollution damages not only the lungs; it also has an impact on skin health and is co-responsible for the development of skin diseases. Anti-pollution products are on the rise in the cosmetic market but so far, there is no established method to directly assess the impact of pollution on the skin and to test the efficacy of anti-pollution products. To address this problem, two different chambers were developed for the reproducible exposure to realistic air pollutant concentrations. One chamber for the exclusive use of excised skin and hair samples, the second chamber for ex vivo and in vivo measurements. Measurements of nicotine next to the investigated skin area allow conclusions to be drawn on the particle concentration to which the skin is exposed. Electron paramagnetic resonance spectroscopy, which enables the detection of free radicals in different systems, was applied to assess the hazard potential of pollution in the skin. A direct proof of the formation of free radicals in the skin by the model pollutant cigarette smoke could be demonstrated. An additional application of UV irradiation even increased the formation of free radicals in the skin seven-fold (sum parameter). Depending on the question of interest, the use of different spin probes allows various assessments of the radical formation in skin: the amount of radicals but also the antioxidant status of the microenvironment can be estimated. Using two exposure chambers, the direct formation of oxidative stress by cigarette smoke on ex vivo skin, with and without additional UV exposure, could be reproducibly examined. This measurement method is promising for the assessment of anti-pollution products and could allow a direct causal connection between pollutant, effect on the skin and the protective function of skin care products.

Multidirectional activity of bakuchiol against cellular mechanisms of facial ageing - Experimental evidence for a holistic treatment approach.

OBJECTIVE: Skin ageing is a multifactorial process involving formation of reactive oxygen species, consecutive inflammation with reduced epidermal and dermal cell viability and resulting damage to the extracellular matrix. Effective dermocosmetic treatment modalities should ideally address these hallmarks in a holistic approach. Here, we determined the corresponding activity profile of bakuchiol, a plant-derived meroterpene, in an array of in vitro, ex vivo and in vivo studies and compared it to retinol, currently considered as gold standard in topical antiageing cosmetics. METHODS: The antioxidative capacity and power of bakuchiol and retinol were analysed by measuring 2,2'-diphenyl-1-picrylhydrazyl (DPPH) reduction via its absorption decay and electron spin resonance spectroscopy, respectively. Effects on prostaglandin E2 (PGE2), macrophage migration inhibitory factor (MIF), fibroblast growth factor 7 (FGF7), collagen type I and VII (COL1A1, COL7A1), fibronectin (FN) levels as well as the metabolization of water-soluble tetrazolium 1 (WST-1) were determined in human dermal fibroblasts. Epidermal regeneration was assessed utilizing an in vitro wound healing model. FN protein levels were analysed ex vivo after treatment with a formulation containing bakuchiol, retinol or vehicle using suction blister fluid. Skin condition improvement was determined in vivo in a split-face comparison study after application of bakuchiol or vehicle. RESULTS: In contrast to retinol, bakuchiol demonstrated high antioxidative efficacy. Levels of PGE2 and MIF were significantly decreased by both bakuchiol and retinol. Bakuchiol but not retinol significantly increased FGF7 protein levels. WST-1 metabolization levels were significantly augmented by bakuchiol and retinol. Bakuchiol and retinol application led to a significant augmentation of COL1A1, COL7A1 and FN protein levels. Wounds supplemented with bakuchiol but not retinol displayed a significant increase in epidermis regeneration. Clinically, areas treated with a bakuchiol-containing formulation showed a statistically significant increase in FN protein values after a 4-week application compared to untreated areas and areas treated with vehicle. CONCLUSION: These data provide evidence for the multidirectional efficacy of bakuchiol against cellular hallmarks of skin ageing. Its activity profile shares some common features with retinol but demonstrates several hitherto unknown biopositive effects in our studies, namely stimulation of the critical extracellular matrix component FN, and accelerated epidermal regeneration and wound healing.

OBJECTIF: le vieillissement de la peau est un processus multifactoriel impliquant la formation de dérivés réactifs de l'oxygène, une inflammation consécutive qui entraîne une viabilité réduite des cellules du derme et de l'épiderme, et endommage la matrice extracellulaire. Pour être efficaces, les traitements dermocosmétiques devraient dans l'idéal traiter ces caractéristiques selon une approche holistique. Ici, nous avons déterminé le profil d'activité correspondant du bakuchiol, un méroterpène d'origine végétale, dans une série d'études in vitro, ex vivo et in vivo, et l'avons comparé au rétinol, qui est aujourd'hui considéré comme la référence parmi les cosmétiques anti-âge topiques. MÉTHODES: la capacité antioxydante et la puissance du bakuchiol et du rétinol ont été analysées en mesurant la réduction du 2,2-diphényl-1-picrylhydrazyl (DPPH) selon sa décroissance par absorption et à l'aide d'une spectroscopie par résonance magnétique électronique, respectivement. Les effets sur la prostaglandine E2 (PGE2), le facteur inhibiteur de la migration (FIM) des macrophages, le facteur de croissance des fibroblastes 7 (FGF7), le collagène de type I et VII (COL1A1, COL7A1), les taux de fibronectine (FN), ainsi que la métabolisation du tétrazolium 1 soluble dans l'eau (WST-1) ont été déterminés dans des fibroblastes dermiques humains. La régénération épidermique a été évaluée à l'aide d'un modèle de cicatrisation des plaies in vitro. Les taux de fibronectine ont été analysés ex vivo après un traitement avec une formulation contenant du bakuchiol, du rétinol ou un excipient à l'aide d'un liquide d'aspiration sous forme de vésicules. L'amélioration de l'état de la peau a été déterminée in vivo dans une étude de comparaison en hémiface après l'application de bakuchiol ou d'un excipient. RÉSULTATS: Contrairement au rétinol, le bakuchiol s'est avéré présenter une efficacité antioxydante élevée. Les taux de PGE2 et de FIM ont significativement diminué avec le bakuchiol et le rétinol. L'application de bakuchiol s'est accompagnée d'une augmentation significative des taux de protéine FGF7, mais pas celle de rétinol. Les taux de métabolisation du WST-1 ont augmenté de façon significative avec le bakuchiol et le rétinol. L'application de bakuchiol et de rétinol a entraîné une augmentation significative des taux de protéines COL1A1, COL7A1 et fibronectine. Les plaies supplémentées en bakuchiol, mais pas en rétinol, ont montré une augmentation significative de la régénération épidermique. Sur le plan clinique, les zones traitées avec une formulation contenant du bakuchiol ont montré une augmentation statistiquement significative des taux de fibronectine après une application de 4 semaines par rapport aux zones non traitées et aux zones traitées avec un excipient. CONCLUSION: ces données fournissent des preuves de l'efficacité multidirectionnelle du bakuchiol contre les caractéristiques cellulaires du vieillissement de la peau. Son profil d'activité partage certaines caractéristiques communes avec le rétinol, mais démontre plusieurs effets biopositifs jusqu'alors inconnus dans nos études : la stimulation de la fibronectine, composante essentielle de la matrice extracellulaire, et une régénération épidermique et une cicatrisation accélérée des plaies.

The human rhabdomyosarcoma cell line TE671--Towards an innovative production platform for glycosylated biopharmaceuticals.

The market of therapeutic glycoproteins (including coagulation factors, antibodies, cytokines and hormones) is one of the profitable, fast-growing and challenging sectors of the biopharmaceutical industry. Although mammalian cell culture is still expensive and technically complex, the ability to produce desired post-translational modifications, in particular glycosylation, is a major issue. Glycans can influence ligand binding, serum half-life as well as biological activity or product immunogenicity. Aiming to establish a novel production platform for recombinant glycoproteins, the human TE671 cell line was investigated. Since the initial analysis of cell membrane proteins showed a promising glycosylation of TE671 cells for biotechnological purposes, we focused on the recombinant expression of two model glycoproteins of therapeutical relevance. The optimization of the cell transfection procedure and serum-free expression succeeded for the human serine protease inhibitor alpha-1-antitrypsin (A1AT) and the hematopoietic cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF). N-glycan analyses of both purified proteins by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry provided first fundamental insights into the TE671 glycosylation potential. Besides protein specific pattern, strong distinctions - in particular for N-glycan fucosylation and sialylation - were observed depending on the medium conditions of the respective TE671 cell cultivations. The cell line's ability to synthesize complex and highly sialylated N-glycan structures has been shown. Our results demonstrate the TE671 cell line as a serious alternative to other existing human expression systems.

Seamless Coupling of Chemical Glycan Release and Labeling for an Accelerated Protein N-Glycan Sample Preparation Workflow.

Analytical methods fr direct quantitative N-glycan analysis require a sequence of sample preparation and clean-up steps that result in reduced glycan recovery. Therefore, we aimed to combine glycan release and labeling steps. Based on the hypothesis that the reaction mechanism for oxidative chemical glycan release comprises a stable glycan isocyanate intermediate, we investigated whether this could be exploited for the in-situ preparation of fluorescent glycan conjugates. ANTS-labeled N-glycans were derived from chicken ovalbumin via an in-situ chemical release/coupling approach and by standard Peptide-N-Glycosidase F (PNGase F) digestion/reductive amination. Synoptic fluorescence-assisted carbohydrate electrophoresis with UV detection (FACE-UV) analysis yielded matching patterns of fluorescent N-glycan bands in the expected electrophoretic mobility range between hexose units GU-5 and GU-11 of the standard. Anthranilamide (2-AB)-glycan conjugates prepared from a test glycoprotein carrying a predominant Core-F glycan gave single predominant peaks in hydrophilic interaction chromatography with fluorescence detection (HILIC-FLD) and electrospray ionization mass spectrometry (ESI-MS) spectra in agreement with sodiated triply charged Core-F-AB conjugates for both the standard and the in-situ coupling methods. The Core-F-AB conjugate prepared by the in-situ coupling approach had a slightly elevated retention time on HILIC-FLD and an ESI-MS m/z peak in line with a urea-bonded glycan-AB conjugate, with closed pyran ring structures on the glycan moiety. Glycan isocyanates intermittently formed during chemical glycan release, which could be utilized to prepare labeled glycan samples directly from glycoproteins and fluorescent dyes bearing a primary amine functional group.

Engineering of CHO Cells for the Production of Recombinant Glycoprotein Vaccines with Xylosylated N-glycans.

Xylose is a general component of O-glycans in mammals. Core-xylosylation of N-glycans is only found in plants and helminth. Consequently, xylosylated N-glycans cause immunological response in humans. We have used the F-protein of the human respiratory syncytial virus (RSV), one of the main causes of respiratory tract infection in infants and elderly, as a model protein for vaccination. The RSV-F protein was expressed in CHO-DG44 cells, which were further modified by co-expression of ß1,2-xylosyltransferase from Nicotiana tabacum. Xylosylation of RSV-F N-glycans was shown by monosaccharide analysis and MALDI-TOF mass spectrometry. In immunogenic studies with a human artificial lymph node model, the engineered RSV-F protein revealed improved vaccination efficacy.

In Vitro Evaluation of Glycoengineered RSV-F in the Human Artificial Lymph Node Reactor.

Subunit vaccines often require adjuvants to elicit sustained immune activity. Here, a method is described to evaluate the efficacy of single vaccine candidates in the preclinical stage based on cytokine and gene expression analysis. As a model, the recombinant human respiratory syncytial virus (RSV) fusion protein (RSV-F) was produced in CHO cells. For comparison, wild-type and glycoengineered, afucosylated RSV-F were established. Both glycoprotein vaccines were tested in a commercial Human Artificial Lymph Node in vitro model (HuALN®). The analysis of six key cytokines in cell culture supernatants showed well-balanced immune responses for the afucosylated RSV-F, while immune response of wild-type RSV-F was more Th1 accentuated. In particular, stronger and specific secretion of interleukin-4 after each round of re-stimulation underlined higher potency and efficacy of the afucosylated vaccine candidate. Comprehensive gene expression analysis by nCounter gene expression assay confirmed the stronger onset of the immunologic reaction in stimulation experiments with the afucosylated vaccine in comparison to wild-type RSV-F and particularly revealed prominent activation of Th17 related genes, innate immunity, and comprehensive activation of humoral immunity. We, therefore, show that our method is suited to distinguish the potency of two vaccine candidates with minor structural differences.

Recombinant glycoproteins: The impact of cell lines and culture conditions on the generation of protein species.

Glycosylation is the most complex post-translational modification. Thus, it contributes to versatile chemical compositions of proteins, leading to high amounts of protein species. The structural heterogeneity of glycoproteins was also described by the definition of glycoforms. We therefore introduced a new term called "glycoprotein species" to join the two concepts from different fields of biology. In this study, we further determined the theoretical numbers of glycoprotein species of two recombinant glycoproteins - a therapeutical antibody and the human protease inhibitor alpha-1-antitrypsin (A1AT) - based on structural analysis of their N-glycans. Moreover, we showed that variations in the used cell lines and their cultivation conditions strongly influence the number of glycoprotein species in case of recombinant A1AT production. BIOLOGICAL SIGNIFICANCE: Protein glycosylation is a major source for the huge amount of protein species. This study extends the sight of protein species by the following contributions: 1) The new term "glycoprotein species" was defined to introduce the concept of glycoforms into the field. 2) An estimation of the number of potential glycoprotein species of two particular glycoproteins was given. 3) The influence of production conditions for recombinant glycoproteins on glycoprotein species generation was displayed.

Oxidation-induced ferritin turnover in microglial cells: role of proteasome.

Highly oxidized protein aggregates accumulating in the brain during neurodegenerative diseases are often surrounded by microglia. Most of the microglial cells surrounding these plaques are activated and release a high amount of oxidizing species. In order to develop their toxic effects numerous oxidizing species need iron. To prevent this iron-dependent oxidation an iron-sequestering apparatus exists, including the major iron storage protein ferritin. Microglial cells damage their own protein pool during activation and it is still unknown whether microglial cells are able to maintain their iron-sequestering function during oxidative stress. Therefore, we explored the microglial cell line RAW to test the maintenance of ferritin under oxidizing conditions. Our investigations revealed a half-life of both ferritin chains of 3-3.5 h and a reduced half-life due to oxidation. This was due to the removal of oxidized ferritin by the proteasomal system. Ferritin de novo synthesis was also severely affected by oxidation. This results in a decreased ferritin pool due to acute oxidative stress. These data let us conclude that microglial cells do not increase their ferritin amount after oxidative stress and an increase in the iron storage capacity in these cells after treatment might be achieved only by a high iron saturation of the existing ferritin molecules.

Increased proteolysis after single-dose exposure with hepatotoxins in HepG2 cells.

Chronic ethanol consumption is associated with increased protein oxidation and decreased proteolysis in the liver. We tested the hypothesis that even single-dose treatment with ethanol or bromotrichloromethane causes increased protein oxidation and a distinct proteolytic response in cultured hepatocytes. HepG2 cells were treated for 30 min with ethanol, H(2)O(2) and bromotrichloromethane at various nontoxic concentrations. Protein degradation was measured in living cells using [35S]-methionine labeling. Protein oxidation, and 20S proteasome activity were measured in cell lysates. Oxidized proteins increased immediately after ethanol, H(2)O(2), and bromotrichloromethane exposure, but a further significant increase 24-h after exposure was observed only following ethanol and bromotrichloromethane treatment. All three reagents caused a significant increase of the overall intracellular proteolysis at rather low concentrations, which could be suppressed by the proteasome inhibitor lactacystin. A decline of proteolysis observed at higher-subtoxic-concentrations was not related to decreased proteasome activity. Preincubation with ketoconazole or 4-methylpyrazole completely prevented the ethanol- and bromotrichloromethane-induced but not the H(2)O(2)-induced protein oxidation and proteolysis, suggesting strongly an enzyme-mediated generation of reactive oxygen species. In conclusion single-dose exposure with ethanol or haloalkanes causes increased protein oxidation followed by an increased proteasome-dependent protein degradation in human liver cells.

Protein oxidation and the degradation of oxidized proteins in the rat oligodendrocyte cell line OLN 93-antioxidative effect of the intracellular spin trapping agent PBN.

Oligodendrocytes are the myelin-producing cells in the central nervous system. It was proposed that these cells are much more prone to oxidative damage than to other cells of the central nervous system. This fact seems to be due to their high iron store and low antioxidative defense mechanisms. Consequently, free radical induced damage should lead to an enhanced damage of oligodendrocytes. Thus, we chose the oligodendrocyte cell line OLN 93 to measure the stability of the protein pool after oxidation and the possibilities of protecting proteins by alpha-phenyl-N-tert-butylnitrone (PBN). We were able to demonstrate for the first time that OLN 93 cells are able to respond with an increase in overall proteolysis when exposed to various oxidants. This increase was the consequence of an enhanced protein oxidation. The activity of the 20S proteasome, which is thought to be involved in the removal of oxidized proteins, was not effected by moderate concentrations of the oxidants. The spin-trap PBN was used as an antioxidant and was able to prevent protein oxidation in OLN 93 cells effectively. Consequently, we proved that PBN is also able to prevent the increase in overall protein oxidation. We were able to demonstrate that OLN 93 oligodendrocytes react to oxidative stress with an increase in the protein turnover directed towards the removal of oxidized proteins. The intracellular spin-trap PBN is able to prevent protein oxidation in OLN 93 cells.

Alpha-lipoic acid prevents ethanol-induced protein oxidation in mouse hippocampal HT22 cells.

Oxidative stress is involved in a number of neurological disorders, including the neurotoxic effects of ethanol. Recent studies have described a neuroprotective potential of alpha-lipoic acid (LC) in several models of neuronal cell death related to oxidative stress. We tested the hypothesis that LC could be effective in preventing ethanol-induced neurotoxicity employing the clonal hippocampa cell line HT22. A 24 h incubation with ethanol 100-600 mM caused a dose-dependent loss of cell viability and a significant increase of the overall intracellular protein oxidation. Coincubation with LC 0.1 mM resulted in a significant decrease of ethanol-related neurotoxicity and a complete prevention of the ethanol-induced intracellular protein oxidation. These results indicate that the radical scavenging properties of LC are effective to ameliorate ethanol-induced neurotoxicity.

Adaptation of human CD4+ T cells to pathophysiological hypoxia: a transcriptome analysis.

OBJECTIVE: Inflamed tissues are usually characterized by low oxygen levels. We investigated whether pathophysiological hypoxia (pO(2) < 1%) as found in the rheumatoid synovium modulates the transcriptome of human CD4+ T cells. METHODS: We analyzed the extent to which hypoxia influences the transcriptome in the rheumatoid synovium according to a gene cluster reflecting adaptation to low oxygen levels. Hypoxia-inducible factor-1alpha (HIF-1alpha) was detected in the rheumatoid synovium using immunohistochemistry. Isolated human CD4+ T cells were exposed to hypoxia and analyzed using microarray analysis, quantitative polymerase chain reaction, and immunoblot detection. RESULTS: In rheumatoid arthritis (RA) synovial tissue samples, hypoxia modulates the transcription profile. This profile is similar, but not identical, to that found in isolated CD4+ T cells incubated under hypoxic conditions. We show that HIF-1alpha is expressed in synovial tissue samples and in hypoxic CD4+ cells; and that hypoxia directly affects differential gene expression in human T cells with up to 4.8% modulation of the transcriptome. Functional genome analysis revealed substantial effects of hypoxia on immune response, transcriptional regulation, protein modification, cell growth and proliferation, and cell metabolism. CONCLUSION: Severe hypoxia, a feature of joint inflammation, considerably modulates the transcriptome of cells found in the rheumatoid synovium. Human CD4+ T cells adapt to hypoxic conditions mainly by HIF-1-driven effects on the transcriptome reflecting a profound influence on immune functions. Thus, hypoxia must be taken into account when therapeutically targeting inflammation.

Antioxidants effectively prevent oxidation-induced protein damage in OLN 93 cells.

Oxidative stress is supposed to play an important role in demyelinating diseases. Oligodendrocytes are the myelin-forming cells in the brain and are highly susceptible to oxidative stress due to their low antioxidative defense systems and high metabolic rate. In the present work, we tested the response of the oligodendrocyte cell line OLN 93 to oxidative stress. OLN 93 cell cultures are characterized by a loss of cell viability after oxidation. This loss of cell viability is accompanied by an increase in protein oxidation and consequently an elevated overall proteolysis. To minimize the oxidative damage, we tested the effects of the antioxidants alpha-lipoic acid and coenzyme Q(10). Both compounds were able to elevate cell viability and to decrease intracellular protein turnover and oxidant induced protein oxidation. Therefore, we concluded that the excessive oxidative damage of oligodendrocytes and their protein pool can be prevented by the usage of antioxidants.

Selective degradation of oxidatively modified protein substrates by the proteasome.

Oxidative stress in mammalian cells is an inevitable consequence of their aerobic metabolism. Oxidants produce modifications to proteins leading to loss of function (or gain of undesirable function) and very often to an enhanced degradation of the oxidized proteins. For several years it has been known that the proteasome is involved in the degradation of oxidized proteins. This review summarizes our knowledge about the recognition of oxidized protein substrates by the proteasome in in vitro systems and its applicability to living cells. The majority of studies in the field agree that the degradation of mildly oxidized proteins is an important function of the proteasomal system. The major recognition motif of the substrates seems to be hydrophobic surface patches that are recognized by the 20S 'core' proteasome. Such hydrophobic surface patches are formed by partial unfolding and exposure of hydrophobic amino acid residues during oxidation. Oxidized proteins appear to be relatively poor substrates for ubiquitination, and the ubiquitination system does not seem to be involved in the recognition or targeting of oxidized proteins. Heavily oxidized proteins appear to first aggregate (new hydrophobic and ionic bonds) and then to form covalent cross-links that make them highly resistant to proteolysis. The inability to degrade extensively oxidized proteins may contribute to the accumulation of protein aggregates during diseases and the aging process.

Standardized extracts of flavonoids increase the viability of PC12 cells treated with hydrogen peroxide: effects on oxidative injury.

Oxidative stress plays an important role in cell death associated with many diseases. In the present study, concentration-dependence of hydrogen peroxide on rat pheochromocytoma (PC12) cell viability was studied. Preventive effects of antioxidants on the viability of these cells treated with 2 mM hydrogen peroxide were compared. Trolox and Stobadine, as chain-breaking antioxidants were studied in comparison with standardized extracts of flavonoids of Ginkgo biloba and Pycnogenol, known as agents effective in several diseases. All antioxidants increased the viability of hydrogen peroxide-treated PC12 cells. Flavonoid extracts were more effective than Trolox and Stobadine. Antioxidants were most effective if present after the oxidative treatment. As expected, the preloading with antioxidants was without effect on cell viability. Correlations between viability increase induced by antioxidants, and content of oxidation products of proteins and lipids were studied at concentrations of antioxidants mostly effective in preventing cell death: Trolox (10 microM), Stobadine (30 microM), Ginkgo biloba (160 microg/ml), Pycnogenol (100 microg/ml). In these concentrations, antioxidants did not statistically significantly decrease the content of protein carbonyls, with exception of Stobadine, which had no effect. Ginkgo biloba, Trolox and Stobadine intensively decreased the content of malondialdehyde, a product of lipid peroxidation. Pycnogenol was without any preventive effect. Concentrations of antioxidants with a large effect on viability of PC12 cells were not effective in preventing oxygen radical-induced injury of proteins. Antioxidants prevented the oxidative injury of lipids more effectively than that of proteins.

Substrate specificity and kinetic properties of seven heterologously expressed dog cytochromes p450.

Seven dog cytochromes p450 (p450s) were heterologously expressed in baculovirus-Sf21 insect cells. Of all enzymes examined, CYP1A1 exhibited high 7-ethoxyresorufin O-deethylase activity (low Km enzyme, 1 microM). CYP2B11 and CYP3A12 effectively catalyzed the N1-demethylation and C3-hydroxylation of diazepam (and its derivatives), whereas CYP3A12 and CYP2D15 catalyzed exclusively the N- and O-demethylation, respectively, of dextromethorphan. However, no saturation velocity curves for the N-demethylation of dextromethorphan (up to 500 microM) were achieved, suggesting a high Km for CYP3A12. In contrast to CYP3A12, the CYP2D15-dependent O-demethylation of dextromethorphan was a low Km process (Km = 0.7 microM), similar to that in dog liver microsomes (Km = 2.3 microM). CYP2D15 was also capable of metabolizing bufuralol (1'-hydroxylation), with a Km of 3.9 microM, consistent with that obtained with dog liver microsomes. CYP3A12 was shown to primarily oxidize testosterone at 16alpha-, 2alpha/2beta-, and 6beta-positions. Selectivity of CYP3A12 was observed toward testosterone 6beta-(Km = 83 microM) and 2alpha/2beta-hydroxylations (Km = 154 microM). However, the 16alpha-hydroxylation of testosterone was catalyzed by CYP2C21 also (Km = 6.4 microM for CYP2C21). Therefore, the 6beta- and 16alpha-hydroxylation of testosterone can potentially be employed as markers of CYP3A12 and CYP2C21 (at low concentration), respectively. CYP2C21 was also capable of catalyzing diclofenac 4'-hydroxylation, although some activity was detected with CYP2B11. Surprisingly, none of the p450s selectively metabolized (S)-mephenytoin 4'-hydroxylation. The results described herein are a first step toward the systematic evaluation of a panel of dog p450s and the development of dog p450 isoenzyme-selective marker substrates, as well as providing useful information on prediction and extrapolation of the results from in vitro to in vivo and from dog to human.