Mercury modulates the cytochrome P450 1a1, 1a2 and 1b1 in C57BL/6J mice: in vivo and in vitro studies

In the current study C57BL/6J mice were injected intraperitoneally with Hg^2 + in the absence and presence of TCDD. After 6 and 24 h the liver was harvested and the expression of Cyps was determined. In vitro, isolated hepatocytes were incubated with TCDD in the presence and absence of Hg^2 +. At the in vivo level, Hg^2 + significantly decreased the TCDD-mediated induction of Cyps at 6 h while potentiating their levels at 24 h. In vitro, Hg^2 + significantly inhibited the TCDD-mediated induction of Cyp1a1 in a concentration- and time-dependent manner. Interestingly, Hg^2 + increased the serum hemoglobin (Hb) levels in mice treated for 24 h. Upon treatment of isolated hepatocytes with Hb alone, there was an increase in the AhR-dependent luciferase activity with a subsequent increase in Cyp1a1 protein and catalytic activity levels. Importantly, when hepatocytes were treated for 2 h with Hg^2 + in the presence of TCDD, then the medium was replaced with new medium containing Hb, there was potentiation of the TCDD-mediated effect. In addition, Hg^2 + increased heme oxygenase-1 (HO-1) mRNA, which coincided with a decrease in the Cyp1a1 activity level. When the competitive HO-1 inhibitor, tin mesoporphyrin was applied to the hepatocytes there was a partial restoration of Hg^2 +-mediated inhibition of Cyp1a1 activity. In conclusion, we demonstrate for the first time that there is a differential modulation of the TCDD-mediated induction of Cyp1a1 by Hg^2 + in C57BL/6J mice livers and isolated hepatocytes. Moreover, this study implicates Hb as an in vivo specific modulator of Cyp1 family.     

Autoinduction of the metabolism of phenothiazine neuroleptics in a primary culture of human hepatocytes

BackgroundThe metabolism of phenothiazine neuroleptics (promazine, perazine) in a primary culture of human hepatocytes after pretreatment of cells with those neuroleptics was studied.MethodsThe hepatocytes were pretreated with 25 μM promazine or perazine for 96 h. Then, the cells were incubated for 2, 4, 6, 8 and 24 h in the presence of neuroleptics. At the indicated time points, concentrations of phenothiazines and their metabolites (5-sulfoxides and N-desmethyl derivatives) were measured in the culture medium using HPLC with UV detection.ResultsPretreatment of the primary culture of human hepatocytes with promazine or perazine resulted in accumulation of their metabolites in the culture medium. Such an effect was not observed in the case of control cultures (not pretreated with neuroleptics).ConclusionThe obtained results suggest that the tested phenothiazines may stimulate their own metabolism by inducing CYP1A2, CYP3A4 and CYP2C19 isoforms.     

Follow-up to the pre-validation of a harmonised protocol for assessment of CYP induction responses in freshly isolated and cryopreserved human hepatocytes with respect to culture format,treatment, positive reference inducers and incubation conditions

We have compared induction responses of human hepatocytes to known inducers of CYP1A2, CYP2B6, CYP2C and CYP3A4/5 to determine whether the culture format, treatment regimen and/or substrate incubation conditions affected the outcome. CYP induction responses to prototypical inducers were equivalent regardless of pre-culture time (24 h or 48 h), plate format (60 mm or 24-well plates) used or whether CYP activities were measured in microsomes or whole cell monolayers. Fold-induction of CYP3A4/5 by 1000 μM PB and 10 μM RIF were equivalent. In contrast, the fold-induction of CYP2B6 by PB was 3-fold higher that by 10 μM RIF. In addition to inducing CYP1A2, 50 μM OME also induced CYP3A4/5 in 50% of the donors tested. CYP2B6 was induced in 14 out of 21 donors by BNF; however CYP3A4/5 was unaffected by BNF in these donors. In order to confirm that donor-to-donor variation was not due to inter-laboratory differences, the induction responses of 5 different batches of cryopreserved human hepatocytes were compared in two different laboratories. The induction of CYP1A2, CYP2B6 and CYP3A4 measured in our laboratory were equivalent to those obtained by the commercial companies, proving good between-laboratory reproducibility.In conclusion, there is some flexibility in the treatment and incubation protocols for classical CYP induction assays on human hepatocytes. Both RIF and PB are suitable positive control inducers of CYP3A4/5 but PB may be more appropriate for CYP2B6 induction. BNF may be more appropriate for CYP1A2 induction than OME since, in contrast to the latter, it does not induce CYP3A4. Induction responses using hepatocytes from the same donor but in different labs can be expected to be similar. The good reproducibility of induction responses between laboratories using cryopreserved hepatocytes underlines the usefulness of these cells for these types of studies.     

Characterisation of the roles of ABCB1, ABCC1, ABCC2 and ABCG2 in the transport and pharmacokinetics of actinomycin D in vitro and in vivo

Actinomycin D plays a key role in the successful treatment of Wilms tumour. However, associated liver toxicities remain a drawback to potentially curative treatment. We have used MDCKII cells over-expressing ABCB1, ABCC1, ABCC2 and ABCG2, alongside knockout mouse models to characterise actinomycin D transport and its impact on pharmacokinetics. Growth inhibition, intracellular accumulation and cellular efflux assays were utilised. A 59-fold difference in GI₅₀ was observed between MDCKII-WT and MDCKII-ABCB1 cells (12.7 nM vs. 745 nM, p < 0.0001). Reduced sensitivity was also seen in MDCKII-ABCC1 and ABCC2 cells (GI₅₀ 25.7 and 40.4 nM respectively, p < 0.0001). Lower intracellular accumulation of actinomycin D was observed in MDCKII-ABCB1 cells as compared to MDCKII-WT (0.98 nM vs. 0.1 nM, p < 0.0001), which was reversed upon ABCB1 inhibition. Lower accumulation was also seen in MDCKII-ABCC1 and ABCC2 cells. Actinomycin D efflux over 2 h was most pronounced in MDCKII-ABCB1 cells, with 5.5-fold lower intracellular levels compared to WT. In vivo studies showed that actinomycin D plasma concentrations were significantly higher in Abcb1a/1b^?/? as compared to WT mice following administration of 0.5 mg/kg actinomycin D (AUC_0–6 h 242 vs. 152 μg/L h respectively). While comparable actinomycin D concentrations were observed in the kidneys and livers of Abcb1a/1b^?/? and Abcc2^?/? mice, concentrations in the brain were significantly higher at 6 h following drug administration in Abcb1a/1b^?/? mice compared to WT. Results confirm actinomycin D as a substrate for ABCB1, ABCC1 and ABCC2, and indicate that Abcb1a/1b and Abcc2 can influence the in vivo disposition of actinomycin D. These data have implications for ongoing clinical pharmacology trials involving children treated with actinomycin D.     

Effect of prenatal manganese intoxication on [3H]glucose uptake in the brain of rats lesioned as neonates with 6-hydroxydopamine

In the present study we examined the effects of prenatal manganese (Mn) intoxication on [³H]glucose uptake in the brain of rats lesioned as neonates with 6-hydroxydopamine (6-OHDA). MnCl₂ ? 4H2O (10,000 ppm) was added to the drinking water of pregnant Wistar rats for the duration of pregnancy. On the day of parturition, Mn was discontinued as an additive to the drinking water. The control group consisted of rats that consumed water without Mn. Three days after birth, rats in both groups (control and Mn) were pretreated with desipramine hydrochloride (20 mg/kg) and pargyline hydrochloride (50 mg/kg) and injected bilaterally icv with one of three doses of 6-OHDAhydrobromide (15 μg, 30 μg or 67 μg base form in saline on each side) or with saline (control). 6-[³H]-Dglucose (500 μCi/kg, ip) was administered to male offspring in adulthood; after 15 min, brain specimens were taken (frontal cortex, hippocampus, striatum, thalamus with hypothalamus, pons and cerebellum) for determination of radioactivity in a liquid scintillation counter. Low dose 6-OHDA (15 μg icv) increased [³H]glucose uptake in all brain regions (p < 0.05) in both control and Mnintoxicated animals. In rats lesioned with a moderate dose of 6-OHDA(30 μg icv), [3H]glucose uptake was unaltered in both control and Mn-exposed rats. High dose 6-OHDA(67 μg icv) reduced [³H]glucose uptake in all brain regions of Mn-exposed rats (except for cerebellum) compared with the saline group (all, p < 0.05). There was no change in regional brain uptake of [³H]glucose in control rats. In conclusion, this study shows that mild neuronal insult (15 μg icv 6-OHDA) increased glucose uptake in the brain while severe damage (concomitant 60 μg icv 6-OHDA and Mn treatment) significantly diminished this process.     

Induction of cytochrome P450 enzymes in primary equine hepatocyte culture

In this study, we established cell culture conditions for primary equine hepatocytes allowing cytochrome P450 enzyme (CYP) induction experiments.Hepatocytes were isolated after a modified method of Bakala et al. (2003) and cultivated on collagen I coated plates. Three different media were compared for their influence on morphology, viability and CYP activity of the hepatocytes. CYP activity was evaluated with the fluorescent substrate 7-benzyloxy-4-trifluoromethylcoumarin. Induction experiments were carried out with rifampicin, dexamethasone or phenobarbital. Concentration–response curves for induction with rifampicin were created.Williams’ medium E showed the best results on morphology and viability of the hepatocytes and was therefore used for the following induction experiments. Cells cultured in Dulbecco’s Modified Eagle Medium were not inducible. Incubation with rifampicin increased the CYP activity in two different hepatocyte preparations in a dose dependent manner (EC₅₀ = 1.20 μM and 6.06 μM; E_max = 4.1- and 3.4-fold induction). No increase in CYP activity was detected after incubation with dexamethasone or phenobarbital.The hepatocyte culture conditions established in this study proved to be valuable for investigation of the induction of equine CYPs. In further studies, other equine drugs can be evaluated for CYP induction with this in vitro system.     

Cytotoxic effects of 109 reference compounds on rat H4IIE and human HepG2 hepatocytes. III: Mechanistic assays on oxygen consumption with MitoXpress and NAD(P)H production with Alamar Blue™

In vitro toxicity screening can reduce the attrition rate of drug candidates in the pharmaceutical industry in the early development process. The focus in this study is to compare the sensitivity for cytotoxicity of a time-resolved fluoro metric oxygen probe with that of a fluoro metric Alamar Blue? (AB) assay. Both assays measure mitochondrial activity by either oxygen consumption (LUX-A65 N-1 (MitoXpress, Luxcel) probe) or NADH/FADH conversion (AB). Both assays were carried out with increasing concentrations of 109 reference compounds using rat H4IIE and human HepG2 hepatocytes at incubation periods of 24, 48 and 72 h. Prior to this study, the influence on medium with either glucose or galactose was studied to analyze the rate of glycolysis and oxygen consumption, which latter process may be impaired in hepatoma cells. Inhibitors of oxygen consumption in combination with a glucose up-take inhibitor showed the largest consumption rate differences in the presence of 5 mM of glucose.The choice for the 109 reference compounds was based on the so-called Multicentre Evaluation for In vitro Cytotoxicity (MEIC) and on diverse drug categories. For 59 toxic reference compounds, an evaluation for both assays was carried up to 10^?3 M. Toxicity was demonstrated with MitoXpress for 23 (39%) and 36 (61%) compounds in H4IIE and HepG2 cells, respectively, and with AB for 44 (75%) and 40 (68%) compounds. For 50 more pharmaceutical drugs more physiological concentrations were used up to 3.16 × 10^?5 M, and only 19 (38%) of these compounds appeared to be toxic in both assays.In conclusion, overall 63 (58%) and 60 (55%) compounds showed toxic effects with the MitoXpress and AB assays on rat H4IIE and human HepG2 hepatocytes, respectively. AB assays were more sensitive with respect to H4IIE cells and MitoXpress assays with respect to HepG2 cells. At all tested time intervals, MitoXpress showed its sensitivity, while AB is more sensitive at 48 and 72 h. With AB more toxic compounds were identified, whereas MitoXpress was more sensitive for a few compounds. A species specific difference was clearly found with digoxin, a human specific potassium channel inhibitor. Thus both assays are valuable identifiers of early toxicity with discrimination in time, compounds and species.     

Effects of topical pimecrolimus 1% on high-dose ultraviolet B-irradiated epidermal Langerhans cells

Some studies reported no changes in the number of epidermal Langerhans cells (LC) that were observed in mice treated with pimecrolimus, and low-dose stimulated solar radiation (once)-induced changers in LC are minimally affected by pimecrolimus. This study is to investigate the effects of topical pimecrolimus 1% on high-dose ultraviolet B (UVB)-irradiated epidermal LC. Forty human foreskin tissues were randomly divided into 4 groups of 10 tissues each: Group A, control; Group B, pimecrolimus 1% (once)-only; Group C, 180 mJ/cm² UVB (once)-only; Group D, UVB + pimecrolimus. Each tissue was cut into 4 pieces corresponding to 4 time points. All the tissues were cultured at 37 °C. After being treated, the tissues were collected respectively and processed for immunohistochemical staining and immunofluorescence staining. For UVB-only group, epidermal CD1a⁺ LC number at 18 h decreased from 39.6 ± 8.30 to 22.3 ± 2.26/5 high magnification, compared to CD1a⁺ LC number at 0 h (P < 0.01). The CD1a⁺ LC number of UVB-only group was significantly less than other groups at 18 h, 24 h and 48 h (P < 0.05, respectively). Similar results were obtained with immunofluorescence staining for CD 1a and immunohistochemical staining for Langerin. The numbers of epidermal HLA-DR⁺ LC had no significant differences among all groups at different time points. Our study found a single 180 mJ/cm² UVB irradiation significantly reduced epidermal LC numbers at 18 h, 24 h and 48 h, however, topical pimecrolimus could reverse these changes. UVB plus pimecrolimus treatment did not affect human LC maturation.     

Toxicity of CuO nanoparticles and Cu ions to tight epithelial cells from Xenopus laevis (A6): Effects on proliferation,cell cycle progression and cell death

Nanoparticles (NPs) have unique chemical and physical properties caused by their small size (1–100 nm) and high surface to volume ratio. This means that the NPs are potentially more toxic than their bulk counterparts. In the present study a cultured epithelial cell line from Xenopus laevis (A6) was used to investigate toxicity of copper (Cu) in 3 different forms; Cu ions (Cu²⁺), CuO NPs (6 nm) and poly-dispersed CuO NPs (100 nm, poly-CuO). Continuous exposures at concentrations of 143–200 μM demonstrated that cytotoxicity differed among the 3 Cu forms tested and that the effects depend on cell state (dividing or differentiated). Dividing cells treated with poly-CuO, CuO NPs (6 nm) or Cu²⁺ showed cell cycle arrest and caused significant increase in cell death via apoptosis after 48 h, 6 and 7 days of treatment, respectively. Treatment with either CuO NPs (6 nm) or Cu²⁺ caused significant decrease in cell proliferation. Treatments of differentiated cells, revealed the same patterns of toxicity for Cu forms tested, but after shorter exposure periods.     

Isolation and cultivation of metabolically competent alveolar epithelial cells from A/J mice

The A/J mouse strain is used in lung cancer studies. To enable mechanistic investigations the isolation and cultivation of alveolar epithelial cells (AECs) is desirable. Based on four different protocols dispase digestion of lung tissue was best and yielded 9.3 ± 1.5 × 10⁶ AECs. Of these 61 ± 13% and 43 ± 5% were positive for AP and NBT staining, respectively. Purification by discontinuous Percoll gradient centrifugation did not change this ratio; however, reduced the total cell yield to 4.4 ± 1.1 × 10⁶ AECs. Flow cytometry of lectin bound AECs determined 91 ± 7% and 87 ± 5% as positive for Helix pomatia and Maclura pomifera to evidence type II pneumocytes. On day 3 in culture the ethoxyresorufin-O-demethylase activity was 251 ± 80 pmol/4 h × 1.5 × 10⁶ and the production of androstenedione proceed at 243.5 ± 344.4 pmol/24 h × 1.5 × 10⁶ AECs.However, 6-α, 6-β and 16-β-hydroxytestosterone were produced about 20-fold less as compared to androstenedione and the production of metabolites depended on the culture media supplemented with 2% mouse serum or 10% FCS. Finally, by RT-PCR expression of CYP genes was confirmed in lung tissue and AECs; a link between testosterone metabolism and CYP2A12, 3A16 and 2B9/10 expression was established. Taken collectively, AECs can be successfully isolated and cultured for six days while retaining metabolic competence.     

Morphological changes and viability of primary cultured human ocular trabecular meshwork cells after exposure to air

PurposeTo investigate the possible toxic effect of air exposure for an in vitro model of primary human ocular trabecular meshwork cells (HTM).MethodsHTM were isolated from five donor eyes and cultivated at 37 °C. After reaching confluence the cells were seeded on two well chamber slides. The chamber slides were turned upside down in a Petri culture dish full of culture medium and filled with air using a 5 ml syringe, starting this way the exposure of the cells to the air. Subsequently they were placed in the incubation chamber at 37 °C. Six groups of HTM cultures were set up: group 1 consisted of samples in which HTM were exposed to air for 30 min, group 2 for 1 h, group 3 for 3 h, group 4 for 6 h, group 5 for 12 h and group 6 for 24 h.ResultsAt 3 h after exposure, the morphology of the cells was still intact, at 6 h few cells appeared deformed and exhibited characteristics of more senescent cells. At 12 h after exposure to air the HTM cells started losing their typical morphology and appeared enlarged and compromised. Viability was superior to 94% in groups 1–3 while for groups 4, 5, 6 it was 82.7%, 39.5% and 12.7% respectively.ConclusionThe toxic effect of air exposure for the studied in vitro model of HTM is not significant for the time period of one to three hours. However it starts reducing viability and alternating morphology 6 h after exposure until the time period of 24 h, where the percentage of living cells is drastically decreased. Therefore, we suggest that the use of an air bubble especially in glaucomatous patients should be applied with caution.     

Combination of the Electro/Fe3+/peroxydisulfate (PDS) process with activated sludge culture for the degradation of sulfamethazine

In this paper, the major factors affecting the degradation and the mineralization of sulfamethazine by Electro/Fe³⁺/peroxydisulfate (PDS) process (e.g. current density, PDS concentration, Fe³⁺ ions concentration and initial sulfamethazine (SMT) concentration) were evaluated. The relevance of this process as a pretreatment prior to activated sludge culture was also examined. Regarding the impact on SMT degradation and mineralization, the obtained results showed that they were significantly enhanced by increasing the current density and the PDS concentrations in the ranges 1–40 mA cm⁻² and from 1 to 10 mM respectively; while they were negatively impacted by an increase of the initial SMT concentration and the Fe³⁺ concentration, from 0.18 to 0.36 mM and from 1 to 4 mM respectively. The optimal operating conditions were therefore 40 mA cm⁻² current density, 10 mM PDS concentrations, 1 mM Fe³⁺, and 0.18 mM SMT. Indeed, under these conditions the degradation of SMT and its mineralization yield were 100% and 83% within 20 min and 180 min respectively. To ensure a significant residual organic content for activated sludge culture after Electro/Fe³⁺/PDS pre-treatment, the biodegradability test and the biological treatment were performed on a solution electrolyzed at 40 mA cm⁻², 10 mM PDS concentrations, 1 mM Fe³⁺, and 0.36 mM SMT. Under these conditions the BOD₅/COD ratio increased from 0.07 to 0.41 within 6 h of electrolysis time. The subsequent biological treatment increased the mineralization yield to 86% after 30 days, confirming the relevance of the proposed combined process.     

Formaldehyde alters triglyceride synthesis and very low-density lipoprotein secretion in a time-dependent manner

Formaldehyde is a common indoor air pollutant that is toxic to the liver. This study aimed to investigate the effects of formaldehyde on triglyceride metabolism in human hepatocellular carcinoma cells (HepG2). Cell viability was detected using a MTT (3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide) assay. Following treatment with different concentrations of formaldehyde for 24 and 48 h, the intra and extra-hepatocellular triglyceride (TG) content was determined using a chemical-enzymatic method; Western blotting was used to detect the levels of fatty acid synthesis and VLDL-related proteins. Our results showed that cell viability significantly decreased after formaldehyde treatment (0.5–12.5 mM, 24/48 h). Extracellular TG levels in the hepatocytes increased after formaldehyde treatment at 0.004 mM–0.1 mM for 24 h. SREBP-1c, ACC, FASN, and MTP, CES3 and DGAT1 proteins increased significantly after 24 h of formaldehyde treatment. Intracellular TG levels decreased for 48 h treatment of formaldehyde. AMPKα increased significantly in all tested groups and p-AMPK increased significantly after 0.1 mM formaldehyde treatment for 48 h. Our results indicated that short–term formaldehyde exposure balances triglyceride metabolism by promoting hepatocellular TG synthesis and VLDL secretion; Long-term formaldehyde disturbs the TG metabolism balance in the hepatocytes.     

Inhibitory effects and mechanism of 25-OH-PPD on glomerular mesangial cell proliferation induced by high glucose

ObjectiveTo investigate the protective effects and potential mechanism of the compound 25-OH-PPD (PPD) on the glomerular mesangial cells (GMC) under high glucose condition.MethodsThe hypertrophic GMC cells were established by DMEM containing glucose and randomly divided into five groups, including the normal control group (Control), the high glucose model group (HG, 25 mmol L⁻¹), the PPD low dose group (1 μmol L⁻¹, PPD-L), the PPD middle dose group (5 μmol L⁻¹, PPD −M) and the PPD high dose group (10 μmol L⁻¹, UCN-H). The GMC were incubated for 48 h under different treatment factors. Total protein content was determined by Lowry method. The diameter of the single GMC and volume were measured by computer photograph analysis system. The GMC cell viability was analyzed by MTT assay. The level of malondialdehyde (MDA), the content of glutathione (GSH) and superoxide dismutase (SOD) activity were measured by ELISA. [Ca²⁺]_і transient was measured by Till image system and by cell-loading Fura-2/AM. The expression of COX-1 and COX-2 were also determined using ELISA method.ResultsThe viability of GMC and the total protein content were decreased in HG group, different dosage PPD group could increase these indexes (P < 0.05). The level of MDA was increased, the content of GSH and SOD was decreased in HG group, while PPD could reduce the MDA and enhance GSH and SOD (P < 0.05). Following treatment with different dosage (PPD-L, PPD-M or PPD-H), the [Ca²⁺]_і transient was reduced (P < 0.05 or P < 0.01). Moreover, the expression of COX-1 was decreased while COX-2 expression was increased in different dosage PPD groups.ConclusionThe protective effects of PPD on GMC from HG-induced hypertrophy may be associated with the inhibition of [Ca²⁺]_і transient and decreasing expression of COX-1 via the oxidative-stress injure pathway.     

Halofuginone suppresses T cell proliferation by blocking proline uptake and inducing cell apoptosis

Inactivation of T cells is a widely used strategy for immunosuppression. Halofuginone (HF) is an antiprotozoal agent for treating parasites in veterinary medicine, and has been demonstrated to inhibit collagen type 1 synthesis, T helper 17 cell differentiation and cytokine production in activated T cells. The present study was designed to examine the biological effects of HF against T cell receptor and interleukin (IL)-2 stimulated T cell proliferation. T cell proliferation in cultured murine splenocytes was determined by methylthiazol tetrazolium assay. Cell apoptosis was mainly determined by fluorescence-activated cell sorting with Annexin-V and 7-aminoactinomycin D staining. Here, we showed that HF significantly suppressed T cell proliferation in naïve splenocyte cultures in response to alloantigen or anti-CD3 antibody (IC₅₀, 2–2.5 nM; P < 0.0001), or in activated T cell cultures in response to IL-2 (IC₅₀, 16 nM; P < 0.0001) in a dose-dependent manner. HF did neither attenuate IL-2 production in anti-CD3 antibody activated T cells nor disrupt STAT5 signaling in IL-2-stimulated T cells, but its anti-T cell proliferation was correlated with an increase in cell apoptosis and a decrease in proline uptake in culture medium. Further experiments showed that proline supplement in cell culture medium significantly prevented HF-mediated suppression of T cell proliferation and cell apoptosis. In conclusion, these data suggest that HF interferes with proline incorporation or uptake, resulting in apoptosis via amino acid starvation response in T cells in the response to antigen/mitogen or IL-2 stimulation.     

HIV protease inhibitors induce metabolic dysfunction in part via increased JNK1/2 pro-inflammatory signaling in L6 cells

Protease inhibitors (PIs), such as atazanavir sulfate and ritonavir, are used clinically to prevent the progression of HIV and are known to induce insulin resistance. To determine whether PI-mediated insulin resistance is induced by activation of pro-inflammatory cascades, L6 skeletal muscle cells were treated ±atazanavir sulfate, ritonavir, or atazanavir sulfate + ritonavir, and ±insulin. Treatment with atazanavir sulfate, ritonavir, or atazanavir sulfate + ritonavir for 24 or 48 h significantly increased basal glucose uptake (P < 0.05) and atazanavir sulfate + ritonavir treatment increased basal glucose uptake significantly more than ritonavir or atazanavir sulfate treatment alone (P < 0.05). Atazanavir sulfate + ritonavir treatment for 48 h completely prevented insulin stimulation of glucose uptake (P > 0.05). When compared to untreated cells, basal palmitate uptake and oxidation was found to be significantly higher in cells treated with PIs alone or in combination (P < 0.05). Prior PI treatment alone or in combination prevented (P > 0.05) the insulin-mediated increase in palmitate uptake and the insulin-mediated decrease in palmitate oxidation observed in the control group. Atazanavir sulfate treatment alone or in combination with ritonavir significantly increased JNK1/2 phosphorylation when compared to the control or ritonavir group (P < 0.05) and this was accompanied by a rise (P < 0.05) in AKT^Ser473 phosphorylation in the basal state. Total JNK1/2 and p38 MAPK protein content and p38 MAPK phosphorylation state were not altered in any of the treatment groups (P > 0.05). Our data indicate that, in muscle cells, PIs induce metabolic dysfunction that is not limited to insulin-sensitive metabolism and that is potentially mediated by a rise in JNK1/2 pro-inflammatory signaling.     

Fluorometric assessment of acetaminophen-induced toxicity in rat hepatocyte spheroids seeded on micro-space cell culture plates

Hepatotoxicity induced by the metabolic activation of drugs is a major concern in drug discovery and development. Three-dimensional (3-D) cultures of hepatocyte spheroids may be superior to monolayer cultures for evaluating drug metabolism and toxicity because hepatocytes in spheroids maintain the expression of various metabolizing enzymes and transporters, such as cytochrome P450 (CYP). In this study, we examined the hepatotoxicity due to metabolic activation of acetaminophen (APAP) using fluorescent indicators of cell viability and intracellular levels of glutathione (GSH) in rat hepatocyte spheroids grown on micro-space cell culture plates. The mRNA expression levels of some drug-metabolizing enzymes were maintained during culture. Additionally, this culture system was compatible with microfluorometric imaging under confocal laser scanning microscopy. APAP induced a decrease in intracellular ATP at 10 mM, which was blocked by the CYP inhibitor 1-aminobenzotriazole (ABT). APAP (10 mM, 24 h) decreased the levels of both intracellular ATP and GSH, and GSH-conjugated APAP (APAP-GSH) were formed. All three effects were blocked by ABT, confirming a contribution of APAP metabolic activation by CYP to spheroid toxicity. Fluorometric imaging of hepatocyte spheroids on micro-space cell culture plates may allow the screening of drug-induced hepatotoxicity during pharmaceutical development.     

Influence of atorvastatin on the pharmacokinetics and pharmacodynamics of glyburide in normal and diabetic rats

Atorvastatin is a selective HMG-CoA reductase competitive inhibitor, used for the treatment of hyperlipidaemia. It is metabolized by CYP 3A4 and 3A5 isoenzymes in liver. It also has moderate inhibition on metabolizing enzymes like CYP 2C9, 2D6 and 3A4. Hence there is more possibility of atorvastatin for inhibition of metabolism of glyburide, by both CYP 2C9 and 3A4. We have studied the effects of atorvastatin on the pharmacodynamics and pharmacokinetics of glyburide in experimental diabetic rats. Atorvastatin (20 mg/kg p.o.) was given to alloxan-induced diabetic rats for 7 consecutive days followed by glyburide (10 mg/kg p.o.). In the rats co-treated with atorvastatin and glyburide, fasting plasma glucose concentration (60.69 ± 5.70%) was further reduced, markedly as compared with glyburide-treated animals. In co-treated group, the pharmacokinetic parameters like clearance (27.83 ± 3.55 l/h) of glyburide was reduced, while peak plasma concentration (18.39 ± 5.29 μg/ml), area under the plasma concentration time curve (120.02 ± 15.17 μg/ml/h) and elimination half-life (4.09 ± 0.50 h) were significantly increased when compared to glyburide alone administered rats. The results of this study revealed that atorvastatin led to the PK/PD changes have been due to glyburide increased bioavailability, decrease volume of distribution, and/or decrease total clearance may be due to the inhibition of cytochrome P450 metobolic system.     

Evaluation of EpiDerm full thickness-300 (EFT-300) as an in vitro model for skin irritation: Studies on aliphatic hydrocarbons

The aim of this study was to understand the skin irritation effects of saturated aliphatic hydrocarbons (HCs), C9–C16, found jet fuels using in vitro 3-dimensional EpiDerm full thickness-300 (EFT-300) skin cultures. The EFT-300 cultures were treated with 2.5 μl of HCs and the culture medium and skin samples were collected at 24 and 48 h to measure the release of various inflammatory biomarkers (IL-1α, IL-6 and IL-8). To validate the in vitro results, in vivo skin irritation studies were carried out in hairless rats by measuring trans epidermal water loss (TEWL) and erythema following un-occlusive dermal exposure of HCs for 72 h. The MTT tissue viability assay results with the EFT-300 tissue show that 2.5 μl/tissue (≈4.1 μl/cm²) of the HCs did not induce any significant changes in the tissue viability for exposure times up to 48 h of exposure. Microscopic observation of the EFT-300 cross-sections indicated that there were no obvious changes in the tissue morphology of the samples at 24 h, but after 48 h of exposure, tridecane, tetradecane and hexadecane produced a slight thickening and disruption of stratum corneum. Dermal exposures of C12–C16 HCs for 24 h significantly increased the expression of IL-1α in the skin as well as in the culture medium. Similarly, dermal exposure of all HCs for 24 h significantly increased the expression of interleukin-6 (IL-6) and IL-8 in the skin as well as in the culture medium in proportion to the HC chain length. As the exposure time increased to 48 h, IL-6 concentrations increased 2-fold compared to the IL-6 values at 24 h. The in vivo skin irritation data also showed that both TEWL and erythema scores increased with increased HCs chain length (C9–C16). In conclusion, the EFT-300 showed that the skin irritation profile of HCs was in the order of C9 ⩽ C10 ⩽ C11 ⩽ C12 < C13 ≈ C14 ≈ C16 and that the tissue was an excellent in vitro model to predict in vivo irritation and to understand the structural activity relationship of HCs.     

Lasting effect of preceding culture conditions on the susceptibility of C6 cells to peroxide-induced oxidative stress

The aim of the present study was to investigate the influence of the maintenance culture conditions on the competence of C6 rat glioma cells to cope with peroxide-induced oxidative stress. C6 cells were maintained either in Ham’s nutrient mixture F-10 supplemented with 15% horse serum and 2.5% foetal bovine serum (FBS) or in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 5% FBS. The differently cultured cells were exposed under identical conditions to hydrogen peroxide (H₂O₂) and cumene hydroperoxide (CHP) in serum-free DMEM. The cells maintained in high serum Ham’s F-10 medium (1) were less sensitive towards the cytotoxic action of both peroxides (EC₅₀-values: H₂O₂: 193 ± 23 μM; CHP: 94 ± 16 μM) than the cells maintained in low serum DMEM (EC₅₀-values: H₂O₂: 51 ± 10 μM; CHP: 27 ± 11 μM), (2) eliminated the peroxides (initial concentration: 100 μM) with higher rates (H₂O₂: 56 ± 5.5 vs. 32 ± 2.7, CHP: 32 ± 6 vs. 3.4 ± 0.6 nmol/min mg protein), (3) contained more glutathione (30 ± 2.5 vs. 14 ± 1.1 nmol/mg protein) and (4) owned a higher glutathione peroxidase activity (28 ± 3.4 vs. 9.5 ± 0.8 mU/mg protein). Glutathione reductase and catalase activities were not affected. These results demonstrate that the preceding culture conditions have a lasting effect on the susceptibility of cultured cells to oxidative stressors like peroxides. As cause for these differences a dissimilar supply of the cells with serum born antioxidants like selenium and α-tocopherol is discussed.