Repellency to ticks (Acari: Ixodidae) of extracts of Nigella sativa (Ranunculaceae) and the anti-inflammatory DogsBestFriend™.

Motivated by observations that the canine anti-inflammatory cream DogsBestFriend™ (DBF) appeared to deter flies, mosquitoes, and ticks from treated animals, repellent efficacy bioassays using four species of ticks were conducted with three extracts of Nigella sativa L. (Ranunculaceae), a constituent of DBF. The DBF cream was tested against nymphs of lone star tick, Amblyomma americanum (L.). In vertical filter paper assays, the three extracts applied at 0.413 mg extract/cm(2) filter paper repelled 96.7-100 % of brown dog tick, Rhipicephalus sanguineus (Latreille) nymphs, whereas, at the same rate, only one extract repelled >90 % A. americanum nymphs. Adult (mixed sexes) American dog ticks, Dermacentor variabilis (Say), required a higher concentration to be repelled effectively; two extracts, applied at 0.827 mg extract/cm(2) filter paper, repelled ≥90 % of the D. variabilis. In contrast, all extracts applied at much lower concentration (0.206 mg extract/cm(2) filter paper) repelled 100 % adult blacklegged ticks, Ixodes scapularis Say (only females tested). Of the two more repellent extracts, one lost most of its activity against A. americanum nymphs in <4 h when applied at 0.827 mg extract/cm(2) filter paper, whereas the other repelled 66.7 % of the nymphs at 192 h after application. At 0.206 mg extract/cm(2) filter paper, one extract was as repellent as deet against A. americanum nymphs. In a vertical bioassay in which nylon organdy was substituted for filter paper, DBF, at the rates of 1.67 and 0.835 mg cream/cm(2), repelled 76.7 and 30.0 % A. americanum nymphs, respectively. These findings indicate that when applied appropriately DBF should afford some protection to canines against tick bites.

Cross-sectional study of hepatic CYP1A and CYP3A enzymes in hybrid striped bass, channel catfish and Nile tilapia following oxytetracycline treatment.

Terramycin for Fish® (oxytetracycline, OTC) is one of three approved drugs for therapeutic treatment of fish in the United States. Nothing is known, however, of the effects of this therapeutic on drug metabolizing enzymes in fish post-treatment. The main purpose of the study was to examine whether the fish CYP1A and CYP3A enzymes would cross-react with antibodies to known mammalian cytochrome P-450 forms (CYP1A1 and CYP3A). Observational feeding studies of OTC effects were conducted in hybrid striped bass, channel catfish and Nile tilapia. Oxytetracycline was mixed into the feed to achieve a daily dose of 82.8 mg per kg body weight at a feeding rate of 1% body weight per day. Hepatic microsomes of each fish were prepared and Western blotting of CYP1A1 and CYP3A4 and enzyme assays of CYP1A2 and CYP3A4 were performed prior to OTC treatment and on post-treatment days 1, 6, 11 and 21. Both goat anti-rat CYP1A1 and rabbit anti-human CYP3A4 showed good cross-reactivity with all three species in this study. All three species exhibited distinct perturbations in one or more of the variables examined on day 1 post-treatment. Immediately following the 10-day medication period, relative liver weight (RLW) of hybrid striped bass was increased 44% and remained elevated through post-treatment day 21. Increased CYP3A4 enzyme activity and protein abundance were noted in channel catfish and Nile tilapia, respectively. This observational approach demonstrated species differences both in control activities and in the timing and extent of hepatic responses to OTC. The unique perturbations of hepatic CYP450 enzymes in different fish species to OTC treatment observed in this study may have relevance for the use of additional antibiotics or other therapeutics used in aquaculture.

Gastric mucosal cell model for estimating relative gastrointestinal toxicity of non-steroidal anti-inflammatory drugs.

The study objective was to characterize the AGS human gastric mucosal cell line as a model for estimating gastrointestinal toxicity of COX-inhibiting compounds. Rofecoxib, celecoxib, nimesulide, ibuprofen, indomethacin, aspirin, salicylic acid, naproxen and acetaminophen were tested for inhibition of COX-2-mediated prostaglandin E2 synthesis in A549 and AGS cells. The IC50 ratio AGS/A549 was calculated as an estimate of the therapeutic index (TI) for gastrointestinal toxicity. Calculated IC50 values of non-steroidal anti-inflammatory drugs (NSAIDs) in A549 cells were in excellent agreement with published values (r = 0.996; P < 0.005). Calcium ionophore induction of arachidonic acid release in AGS cells provided TI similar to those using platelets and A549 cells (r = 0.918; P < 0.01). The AGS/A549 model exhibited lower TI than the platelet/A549 model. Spearman ranking correlated clinical NSAID gastropathy with lower AGS TI values. The AGS cell line has excellent potential to serve as a model for assessing the gastrointestinal effects of COX-inhibiting compounds.

Serum pharmacokinetics and tissue and milk residues of oxytetracycline in goats following a single intramuscular injection of a long-acting preparation and milk residues following a single subcutaneous injection.

Separate groups of goats were used to determine drug depletion patterns in serum (n=10), tissue (n=20) and milk (n=8) following a single intramuscular (i.m.) dose of 20 mg/kg of a long-acting oxytetracycline (OTC) formulation (Liquamycin LA-200). Milk residues were also determined following a subcutaneous (s.c.) administration of the same product at the same dose. Serum samples were taken for 24 h post-treatment and tissues (fat, liver, kidney, muscle and injection site) collected at 4, 7, 14, 21 and 28 days following injection. Milk from lactating goats was collected every 12 h for 8 days following both the i.m. and s.c. treatments utilizing an intervening 5-week washout period. Residues in serum and tissue were measured using a microbial inhibition assay, while milk residues were measured using both a microbial inhibition assay and a validated HPLC method. The serum pharmacokinetic parameters of OTC in goats were determined, with a mean AUC=67.4 microg h/mL, mean terminal half-life=14.4 h, and apparent clearance=0.33 L/kg h. Tissue half-lives could not be determined with confidence because the collection times provided only two points at which residues could be measured for most tissues. Oxytetracycline residues in all goat tissue samples measured less then cattle tissue tolerance by 96 h postdosing. One-compartment model describing milk depletion data for i.m. and s.c. dosing had terminal slope half-lives of 20.1 and 36.1 h, respectively. By 96 h post-treatment none of the milk samples contained OTC residues in excess of the cattle milk tolerance (0.3 p.p.m.). For both milk and tissue, the upper-bound 99% confidence intervals for the samples taken from goats 96 h postdosing were lower than approved cow milk and tissue tolerances.

A phase I trial of andrographolide in HIV positive patients and normal volunteers.

A phase I dose-escalating clinical trial of andrographolide from Andrographis paniculata was conducted in 13 HIV positive patients and five HIV uninfected, healthy volunteers. The objectives were primarily to assess safety and tolerability and secondarily to assess effects on plasma virion HIV-1 RNA levels and CD4(+) lymphocyte levels. No subjects used antiretroviral medications during the trial. Those with liver or renal abnormalities were excluded. The planned regimen was 5 mg/kg bodyweight for 3 weeks, escalating to 10 mg/kg bodyweight for 3 weeks, and to 20 mg/kg bodyweight for a final 3 weeks. The trial was interrupted at 6 weeks due to adverse events including an anaphylactic reaction in one patient. All adverse events had resolved by the end of observation. A significant rise in the mean CD4(+) lymphocyte level of HIV subjects occurred after administration of 10 mg/kg andrographolide (from a baseline of 405 cells/mm(3) to 501 cells/mm(3); p = 0.002). There were no statistically significant changes in mean plasma HIV-1 RNA levels throughout the trial. Andrographolide may inhibit HIV-induced cell cycle dysregulation, leading to a rise in CD4(+) lymphocyte levels in HIV-1 infected individuals.

Time course comparison of cell-cycle protein expression following partial hepatectomy and WY14,643-induced hepatic cell proliferation in F344 rats.

During recent years, there has been an extensive research focus in the area of cell-cycle control in eukaryotes and the relationship that exists between cell proliferation and cancer. The eukaryotic cell-cycle is governed by signal transduction pathways mediated by complexes of cyclin dependent kinases (CDK) and their partner cyclin proteins. This study was performed to identify differences in cell-cycle control protein expression following physical and chemical stimuli of hepatic cell growth. Protein levels of cell cycle mediators, cyclin dependent kinases (CDK 1,2,4,5), cyclin proteins (A,B,D1-D3 and E), proliferating cell nuclear antigen (PCNA), tumor suppressor proteins (p53 and Rb), and CDK inhibitory proteins (p16Ink4, p21Waf1 and p27Kip1) were examined in F344 rats following 70% partial hepatectomy or a single dose of WY14,643 over 96- and 48-h time courses, respectively. CDK1 (p34cdc2) and PCNA protein concentrations, quantified by ELISA, were significantly increased beginning at the 24-h time point and maximal at 48 h (6.9- and 3.7-fold for partial hepatectomy and 4.2- and 3.3-fold for WY14,643, respectively). Differential effects were observed with the G1 cell-cycle mediators CDK4, CDK5, and cyclin D3, p21Waf1 and p27Kip1 CDK inhibitory protein concentrations rose in accordance with the induction of DNA synthesis and histone H1 kinase activity. In addition, there were dramatic differences in p53 protein expression patterns following partial hepatectomy versus WY14,643 dosing. Because non-genotoxic hepatocarcinogens are known to induce cellular proliferation, data generated from this study may aid in elucidating the specific hepatocarcinogenic signal transduction pathways stimulated by non-genotoxic carcinogens.

Multiple route and dose pharmacokinetics of enrofloxacin in juvenile Atlantic salmon.

The fluoroquinolone antibacterial family is a relatively recent group of bactericidal compounds, generally characterized by efficacy against a wide spectrum of bacterial organisms and exhibiting minimal adverse effects in treated patients. The fluoroquinolones are widely prescribed in both human and veterinary medicine, though in veterinary medicine in the USA there are currently only two approved compounds, enrofloxacin (Baytril, Bayer Animal Health, Shawnee Mission, KS) and sarafloxacin (SaraFlox, Abbott Laboratories, North Chicago, IL), both with limited species and disease label approvals. Currently, there are no approved fluoroquinolone antibacterials to treat bacterial infectious diseases in cultured fish species. Enrofloxacin was administered to juvenile Atlantic salmon as a single bolus via intraarterial (i.a.), intraperitoneal (i.p.), intramuscular (i.m.), or oral gavage routes of administration. The drug was administered via the first three routes to achieve a dose of 10 mg/kg, and via oral gavage to achieve both 10 (p.o.-10) and 5 (p.o.-5) mg/kg doses. Two-compartment model kinetics were observed with elimination of half-lives (t1/2) of 130.6, 34.32, 84.98, 105.11, and 48.24 h, area under the drug concentration-time curves (AUC) of 84.3, 75.31, 55.61, 41.68, and 38.81 micrograms x h/mL, and bioavailabilities (F) of 100, 89.34, 65.97, 49.44, and 46.04% (i.a., i.p., i.m., p.o.-10, p.o.-5, respectively). All administration routes at 10 mg/kg were found to yield comparable drug concentration-time curves for multiple tissue, indicating no distinct advantage of using one route over another from a kinetics perspective. Finally, the 5 mg/kg dose (p.o.-5) yielded comparable multiple tissue drug concentration-time curves to the 10 mg/kg dose (p.o.-10), providing pharmacokinetic evidence to justify therapeutic efficacy trials with the lower dose.

Murine hepatic p53, RB, and CDK inhibitory protein expression following acute 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure.

This study examined the expression of murine hepatic tumor suppressor and cell cycle inhibitory proteins in response to acute 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) dosing in Balb/c mice. Elevations in expression of p53, retinoblastoma (Rb) protein, p16Ink4, p21Waf1 and p27Kip1 were observed six days after a single dose of 0.25, 0.5, 1 or 2 micrograms TCDD/kg. These data suggest that the TCDD-induced inhibition of hepatocyte proliferation in vivo could be attributed to the expression of cell cycle inhibitory proteins.

Discordant hepatic expression of the cell division control enzyme p34cdc2 kinase, proliferating cell nuclear antigen, p53 tumor suppressor protein, and p21Waf1 cyclin-dependent kinase inhibitory protein after WY14,643 ([4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid) dosing to rats.

The hepatocarcinogen and peroxisome proliferator WY14,643 ([4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid) was examined for its ability to induce changes in the intracellular protein expression of hepatic p34cdc2 kinase (CDK1), proliferating cell nuclear antigen (PCNA), p53 tumor suppressor protein, and p21Waf1 CDK inhibiting protein. Young adult male rats were administered 45 mg-kg/day WY14,643 intraperitoneally for 1, 2, 3, 4, or 5 days or fed diets containing 0% or 0.08% WY14,643 for 1, 2, 3, or 4 weeks. WY14,643 dosing increased concentrations of hepatic proteins of 34- and 37-kDa molecular mass, which were identified through immunoprecipitation as CDK1 and PCNA, respectively. Gel filtration of the hepatic S9 fractions determined by enzyme-linked immunosorbent assay confirmed the increased expression of CDK1 and PCNA immunoreactivity in livers from WY14,643-treated rats. Also, gel filtration revealed that the native CDK1 and PCNA in hepatic S9 from WY14,643-treated rats chromatographed as a major peak with an apparent molecular mass of 70 and 76 kDa, respectively. Immunoblotting of the 70-kDa fraction with anti-CDK1 revealed a single band of molecular mass of 34 kDa. Thus, the CDK1 in the major immunoreactive peak of WY14,643-treated rat liver S9 seems to exist as a heterodimer or homodimer. Immunohistochemistry of formalin-fixed liver demonstrated a cytosolic localization of immunoreactive CDK1 and nuclear localization of immunoreactive PCNA in proliferating cells of WY14,643-treated rat livers. WY14,643 increased hepatic CDK1 content by 1.9-6.3-fold through postdosing days 1-5. Hepatic PCNA content was increased 1.9-5-fold over the same period. In the 4-week feeding study, CDK1 and PCNA expression were increased at all weekly time points by an average of 15-50-fold, respectively. Furthermore, the dietary administration of 0.08% WY14,643 resulted in sustained, overexpression of hepatic p53 tumor suppressor protein from week 1 through week 4 and of p21Waf1 CDK inhibitory protein from week 3 to week 4.

Discordant expression of the cyclin-dependent kinases and cyclins in rat liver following acute administration of the hepatocarcinogen [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid (WY14,643).

Cellular proliferation is an essential aspect of chemical carcinogenesis. At the core of cell cycle regulation is a family of serine/threonine protein kinases termed cyclin-dependent kinases (cdk). Cdk activity, which directs progression through the cell cycle, is dependent upon cdk binding to the appropriate, phase-specific cyclin proteins. Alterations in hepatic cdk1, cdk2, cdk4, cdk5, and cyclin protein expression were determined in response to acute dosing of the prototypic peroxisome proliferator and hepatocarcinogen [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid (WY14,643). Intraperitoneal dosing of 45 mg WY14,643/kg daily for 4 days to young, male rats produced dramatic increases in hepatic protein expression of all cdk analyzed as well as cyclins B, D2, D3, and proliferating cell nuclear antigen (PCNA). The largest relative increases, 6.1-, 2.8-, 11-, 83-, and 7.9-fold, were seen with cdk1, cdk4, cyclin B, cyclin D3, and PCNA, respectively. Increases of only 1.8-, 2-, 1.6-, and 1.4-fold were noted, respectively, for cdk2, cdk5, cyclin D2, and cyclin E. Analysis of gel filtration fractions indicated that PCNA co-eluted with cdk1 from the WY14,643-treated rats as a 70-80 kDa molecular complex. In contrast, cdk4, cdk5 and D cyclins migrated as much larger complexes with an estimated MW of approximately 180-190 kDa.

In vitro and in vivo study of the effects of enrofloxacin on hepatic cytochrome P-450. Potential for drug interactions.

Enrofloxacin (EF; BAYTRIL, Miles) was the first fluoroquinolone antimicrobial to be used in veterinary medicine in the US. In humans, fluoroquinolones hinder the metabolism of other clinically important drugs through inhibition of hepatic cytochrome P-450's (P450). Similar interactions are suspected in animals. In this study, we characterized the ability of EF to modify the enzymatic activity of the P450 IA and IIB families. In an in vitro experiment, the inhibition of P450 reductase by EF was assessed by measuring the NADPH-cytochrome c reductase activity, and the inhibition of P450IA1, IA2 and IIB by 0.25, 0.5 and 1.0 mM EF was studied, respectively, by measuring the ethoxy (EROD), methoxy (MROD) and pentoxy (PROD) O-dealkylation activities in rat liver microsomes. NADPH-cytochrome c reductase was not affected. Enrofloxacin induced a strong, concentration-dependent inhibition of P450IA1 and IA2. In an in vivo experiment, the effects of 5 administrations of 5 (EF5), 25 (EF25) or 100 (EF100) mg/kg/d were assessed in rats. The liver cytochrome b5 and total P450 content was assayed by spectrophotometric measurements; P450IA and P450IIB isozyme contents were evaluated by immunoblotting with isozyme specific monoclonal antibodies, and by measuring MROD, EROD and PROD activities. A slight induction of P450IIB1 and IIB2 expression and activity (140% of controls) was only present after EF5 treatment. We concluded that EF directly inhibits P450IA1 and IA2 and advise caution when drugs metabolized extensively by these P450 isozymes are administered in association with EF. The slight stimulation of the P450IIB subfamily is not a concern at the recommended therapeutic dose of 5 mg EF/kg.

A preliminary physiologically based pharmacokinetic model for naphthalene and naphthalene oxide in mice and rats.

Naphthalene is a toxicant with unusual species and tissue specificity that has been the subject of in vitro studies. We describe a preliminary physiologically based pharmacokinetic (PBPK) model for naphthalene constructed solely from in vitro data for comparison to animal data without the use of adjustable parameters. The prototypical PBPK model containing five lumped tissue compartments was developed to describe the uptake and metabolism of naphthalene by mice and rats dosed intraperitoneally (i.p.) and orally (po). The model incorporates circulation and biotransformation of the semistable reactive intermediate, naphthalene oxide, as well as the parent compound naphthalene. Circulation is included because the toxic action of naphthalene has been proposed to be caused by the formation of a reactive metabolite in one organ (liver) and its circulation to another organ (lung) being adversely affected by the metabolite. The model allows conversion of naphthalene oxide into dihydrodiol, glutathione (GSH) conjugates, 1-naphthol (non-enzymatically) and covalently bound adducts with proteins. Model simulations are compared with previously reported in vivo measurements of glutathione depletion, mercapturic acid formation, and covalently bound protein formation. The mouse model predicts accurately the amount of mercapturates excreted, the effect of various pretreatments, and the extent of covalent binding in the lung and liver resulting from ip administration, including the sharp increase in binding between 200 and 400 mg/kg.

Comparison of acute hepatocellular proliferating cell nuclear antigen labeling indices and growth fractions, p34cdc2 kinases, and serum enzymes in carbon tetrachloride-treated rats.

We evaluated various biomarkers associated with cell proliferation immediately following insult with the classic hepatotoxicant carbon tetrachloride (CCl4). Rats were administered a single necrogenic dose of CCl4 and euthanized at either t = 4, 8, 12, 16, or 24 hr postdose. Parameters evaluated included the following: immunohistochemical detection of hepatocellular proliferating cell nuclear antigen labeling indices (PCNA-LIs; percentage of cells in S phase) and growth fractions (PCNA-GFs; percentage of cells in the cell cycle); PCNA and the cyclin-dependent kinase p34cdc2 (CDK) protein in S-9 fractions by Western blot and enzyme-linked immunosorbent assay (ELISA); and liver-related serum enzymes. An increase in PCNA-GF was observed at t = 4 hr, concomitant with elevations in CDK and PCNA protein (Western blot). PCNA-LIs were increased by t = 24 hr, as were CDK and PCNA by ELISA. Sorbitol dehydrogenase was the most sensitive enzyme, with increases observed at t = 4 hr. Our results indicate that PCNA-GF, CDK, and PCNA levels reflect hepatocellular regeneration as early as 4 hr following CCl4 insult. We conclude that these assays are early and sensitive indicators of acute hepatotoxicity that may be advantageous to evaluate in the early stages of exploratory studies.

Pharmacokinetic properties of bromide in dogs after the intravenous and oral administration of single doses.

Bromide (20 mg kg-1) was administered intravenously and orally to normal beagle dogs. The mean (SD) apparent elimination half life (t1/2 beta) after oral administration (46 +/- 9 days) was not significantly different from the mean t1/2 beta after intravenous administration (37 +/- 10 days). The mean total body clearance was 9.0 +/- 3.9 ml day-1 kg-1 and the mean apparent volume of distribution was 0.45 +/- 0.07 litre kg-1. The mean area under the serum concentration time curve (AUC) was significantly smaller after oral administration than after intravenous administration, and from a comparison of the two values the oral bioavailability of bromide was estimated to be 46 per cent. Assuming this degree of bioavailability, the daily dose of bromide necessary to maintain serum bromide concentrations within the therapeutic range of 1000 to 2000 mg litre-1 recommended for epileptic dogs was estimated to be approximately 21 mg kg-1. The intravenous loading dose of sodium bromide necessary to reach minimal therapeutic serum bromide concentrations was predicted to be 570 +/- 90 mg kg-1.

Effect of dietary chloride content on the elimination of bromide by dogs.

The effect of dietary chloride content (0.2, 0.4 and 1.3 per cent chloride on a dry matter basis) on the disposition of a single oral dose of bromide (14 mg kg-1) was evaluated in normal beagles. Increasing the dietary chloride content from 0.2 to 1.3 per cent resulted in a significant decrease in the mean apparent elimination half-life from 69 +/- 22 days to 24 +/- 7 days. The mean area under the concentration curve (AUC) for dogs fed 1.3 per cent chloride was significantly smaller than the AUC for dogs fed 0.2 per cent chloride. Dietary chloride had no effect on the maximum serum concentrations (Cmax) or on the time (Tmax) to reach the maximum concentrations. The steady-state serum bromide concentrations predicted from the single dose data for daily doses of 14 mg kg-1 of bromide were significantly lower in dogs fed 1.3 per cent chloride (310 +/- 150 mg litre-1) than in dogs fed 0.2 per cent chloride (1950 +/- 1140 mg litre-1). The predicted mean daily doses of bromide necessary to maintain serum levels within the therapeutic range for dogs fed 1.3 per cent chloride (43 +/- 13 mg kg-1) were almost twice as high as the dose estimated for dogs fed 0.4 per cent chloride (22 +/- 3 mg kg-1) and nearly three times as high as the dose estimated for dogs fed 0.2 per cent chloride (15 +/- 4 mg kg-1). These differences were statistically significant (P = 0.002).

Possible involvement of the Ah receptor in the induction of cytochrome P-450IA1 under conditions of hydrodynamic shear in microcarrier-attached hepatoma cell lines.

The exposure of two hepatoma cell lines, Hep G2 and Hepa-1, to moderate hydrodynamic shear, in microcarrier-attached suspension cultures, resulted in the transient induction of cytochrome P450IA1 (CYP1A1). Both cell lines have been characterized with respect to their Ah receptor (AhR) concentrations and induce CYP1A1 in response to exposure to xenobiotics such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Using an AhR antagonist, alpha-naphthoflavone (alpha-NF) and a protein kinase C (PKC) inhibitor, staurosporine (ST), in the Hep G2 cell line, the induced CYP1A1 activity was modulated in the same manner as when the cells were coexposed to TCDD and either alpha-NF or ST. Exposure of the Hep G2 cell line to TCDD and shear resulted in both enhancement of the induced CYP1A1 activity in addition to a competitive response. Finally, using the wild type and AhR defective Hepa-1 cell lines, it was demonstrated that a functional AhR was required for shear-induced CYP1A1 expression. The data obtained in the three cell lines indicate a role for the AhR in the induction of CYP1A1 by shear in agitated microcarrier cultures.

A cell culture analogue of rodent physiology: Application to naphthalene toxicology.

The difficulties of large-scale animal testing of compounds has spurred development of in vitro testing methods and physiologically based pharmacokinetic models (PBPK). In existing in vitro methods, tissue interactions occurring in vivo are not reproduced accurately and in PBPKs the a priori prediction of metabolism is difficult. Through development of a multicompartmental, multiple cell type bioreactor system these limitations can be circumvented. A cell culture analogue (CCA) of a PBPK was developed. The CCA contains multiple chambers, each of which represents a tissue or group of similar tissues as specified in the PBPK. Proof-of-concept experiments were done using naphthalene as a model. Naphthalene is converted into naphthalene oxide and the circulation of this reactive metabolite from the liver to lung is a possible mechanism for lung injury. A CCA with liver, lung and other tissue compartments was constructed. This system was used in conjunction with cultured H4IIE rat hepatoma cells and L2 rat lung cells to study the importance of circulated naphthalene metabolites (presumably naphthalene oxides) on lung cell toxicity in rodents. By increasing the number of cells and/or inducing cytochrome P-450 activity in the liver compartment, lung cell mortality was increased. Glutathione depletion in the lung and liver cells was also observed. These results indicate that the CCA is a potentially useful concept for studying the action of compounds with reactive metabolites.

Dose-response relationships in mice following subchronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin: CYP1A1, CYP1A2, estrogen receptor, and protein tyrosine phosphorylation.

The dose-response relationships for different endpoints in different tissues were compared in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment. TCDD was administered 5 days a week for 13 weeks at doses ranging from 1.5 to 150 ng/kg/day to female B6C3F1 mice. Ethoxyresorufin-O-deethylase (EROD) activity, a marker for CYP1A1, was increased in liver, lung, and skin at doses as low as 1.5 ng/kg/day. EROD activity did not attain maximal induction. Liver acetanilide-4-hydroxylase activity, a marker for CYP1A2, was significantly induced at 1.5 ng/kg/day and reached maximal induction at 45 ng/kg/day. TCDD treatment significantly increased the amount of phosphorylated forms of three phosphotyrosyl proteins (pp32, pp34, and pp38) in liver S-20 fractions. Changes in these phosphotyrosyl proteins occurred at 1.5 ng/kg/day and reached maximal induction at 4.5 ng/kg/day. No changes in phosphotyrosyl proteins were observed in skin. Hepatic and uterine estrogen receptor levels were not altered at any of the doses tested. These data indicate that induction of CYP1A1, CYP1A2, and the increases in phosphorylated forms of pp32, pp34, and pp38 are sensitive indicators of TCDD exposure. The dose-response curves for increases in CYP1A1, CYP1A2, and phosphorylated pp32, pp34, and pp38 in liver were different from each other. TCDD produces multiple effects with multiple dose-response curves suggesting that there are events in addition to receptor binding that are endpoint specific, leading to different dose-response relationships.

Acute 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure results in enhanced tyrosylphosphorylation and expression of murine hepatic cyclin dependent kinases.

An increase in tyrosine phosphorylation of two hepatic S9 proteins migrating at 34 and 33 kDa that cross-reacted with anti-PSTAIR antibody on immunoblots was seen 24 h after administration of a single dose of 0.25, 0.5, 1 or 2 micrograms 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)/kg to C57BL/6J female mice. Two hepatic S9 proteins migrating at 34 and 33 kDa that cross-reacted with anti-cdc2 C-terminus antibody on immunoblots were observed in corn oil control mice; increased expression of these proteins was seen with increasing doses of TCDD. A maximal increase in expression of 3-times the control was observed at 1 and 2 micrograms TCDD/kg for both p34 and p33. The stimulation of enhanced tyrosylphosphorylation and expression of cyclin dependent kinases p34cdc2 and p3cdk2 by TCDD is consistent with a mechanism of action of TCDD toxicity associated with stimulation of cellular proliferation.

Effects of ciprofloxacin and enrofloxacin on zoxazolamine kinetics, plasma concentration and sleeping times in mice.

The treatment of CD1 male mice with either ciprofloxacin (CP) or enrofloxacin (EF) prior to zoxazolamine (ZX) administration increased the mean ZX sleeping times to, respectively, 162 and 156% of the control (ZX alone). At the end of the sleeping time, the mean ZX plasma concentration in controls was 27.2 micrograms/ml and was not different in EF- or CP-treated groups (87% and 95% of controls, respectively). The animals coadministered with CP or EF and ZX eliminated the latter more slowly than the controls. The estimated zero-time drug concentration of the disposition curves of both the CP- and EF-treated groups as well as the apparent half-life of elimination and apparent overall rate of elimination of the CP-treated group were different from the control values.