Hydrolysis of Several Glycol Ether Acetates and Acrylate Esters by Nasal Mucosal Carboxylesterase in vitro

Hydrolysis of Several Glycol Ether Acetates and Acrylate Estersby Nasal Mucosal Carboxylesterase in vitro. STOTT, W. T., ANDMCKENNA, M. J. (1985) Fundam. Appl. Toxicol. 5, 399–404.The in vitro activity of carboxylesterase recovered from thenasal mucosal tissue of B6C3F1/Cr1BR mice toward several agentsknown to cause olfactory epithelial lesions when inhaled byrodents was determined. Apparent V_max and K_m values were obtainedfor mouse nasal carboxylesterase using ethylene glycol monomethylether acetate (EGMEAc), ethylene glycol monoethyl ether acetate(EGEEAc), propylene glycol monomethyl ether acetate (PGMEAc),methyl acrylate (ME), ethyl acrylate (EA), and butyl acrylate(BA) as substrates. The short straight-chained glycol ethers,EGMEAc and EGEEAc, appeared to be relatively good substratesfor nasal carboxylesterase under enzyme saturating and subsaturatingconditions as indicated by their high V_max and V_m values. Theshort-chained acrylate esters MA and EA were also hydrolyzedto a greater extent than BA at enzyme-saturating levels; however,the reverse was true at subsaturating levels as indicated bythe relatively high V_max/K_m ratio obtained for BA. MA and BAwere observed to cause a loss of carboxylesterase activity atenzyme saturation levels while EA caused a loss of enzyme activityat only one-half K_m concentration. Using EGMEAc as a substrate,no sex differences in nasal carboxylesterase activity were observedin mice or rabbits. The specific activity of nasal carboxylesterasewas found to be equivalent to that of the liver and greaterthan that of the kidney, lung, or blood. Mice and dogs werefound to have similar nasal carboxylesterase activities whichwere slightly higher than that found in rats and about six-foldhigher than that found in rabbits. These data suggest that extensivehydrolysis of several inhaled glycol ether and acrylate estersoccurs in the nasal mucosa of animals exposed to these materials.This hydrolysis may result in the production of acidic metabolitescapable of causing lesions of the nasal olfactory epitheliumas well as influence the systemic concentrations of these esterswhich are attained in exposed animals.     

Comparative Toxicokinetics of Methanol in the Female Mouse and Rat

The toxicokinetics of methanol in female CD-1 mice and Sprague-Dawleyrats were examined to explore the possibility of species differencesin the disposition of the compound. Mice received a single doseof 2.5 g/kg methanol either po (by gavage) or iv (as a 1-mminfusion). Rats received a single oral dose of 2.5 g/kg methanol.As expected, the disposition of methanol was nonlinear in bothspecies. Data obtained after iv administration of methanol tomice were well described by a one-compartment model with Michaelis-Mentenelimination. Blood methanol concentration-time data after oraladministration could be described by a one compartment (mice)or two-compartment (rats) model with Michaelis-Menten eliminationfrom the central compartment and biphasic absorption from thegastrointestinal tract Kinetic parameters (V_max for elimination,apparent volume of the central compartment [V_e first-order rateconstants for intercompartmental transfer [k₁₂ and k₂₁ and first-orderabsorption rate constants for fast [k_AF] and slow [k_AS] absorptionprocesses) were compared between species. When normalized forbody weight, mice evidenced a higher maximal elimination ratethan rats (V_max=117±3 mg/hr/kg vs 60.7±1.4 mg/hr/kgfor rats). The contribution of the fast absorption process tooverall methanol absorption also was larger in the mouse thanin the rat.     

Toxicokinetics of Intravenous Methanol in the Female Rat

The toxicokinetics of intravenously administered methanol wereexamined in female Sprague-Dawley rats. Animals received a singleadministration of 100, 500, or 2500 mg methanol/kg; the twolower doses were administered as a bolus, while the high dosewas administered over 1.5 min. A small (approximately 3%) butstatistically insignificant (p>0.1) degree of transpulmonarymethanol extraction, expressed as the fractional arterial-venousdifference in concentration, was observed after administrationof 250 mg methanol/kg. The elimination of methanol from thesystemic circulation was markedly nonlinear, suggestive of asignificant capacity-limited route of elimination. A singleset of kinetic parameters (apparent distributional volume ofthe central compartment [V_c], intercompartmental transfer rateconstants [k₁₂ and k₂₁], and V_max and K_m for elimination) describedthe blood methanol concentration-time data from rats receivingthe 100 and 500 mg/kg doses. Blood methanol concentrations declinedmuch more rapidly in animals receiving the 2500 mg/kg dose thanwould be predicted from the kinetic parameters derived fromthe other two experimental groups. The data from the 2500 mg/kggroup could be described adequately by a kinetic model incorporatingparallel first-order and saturable elimination processes. Aportion of this apparent linear elimination pathway was dueto renal excretion of the unchanged alcohol. The presence ofboth linear and nonlinear elimination pathways for methanolmay have implications regarding high-dose to low-dose toxicologicextrapolations.     

关附甲素对豚鼠乳头肌动作电位最大除极速度的频率依赖性抑制作用

应用标准微电极技术，研究了关附甲素对豚鼠右心室乳头肌动作电位最大除极速率(V_max)的频率依赖性抑制作用(RDB)，并与美西律，奎尼丁，劳卡尼进行了比较. 在相同刺激间隔(300 ms)，产生50%左右RDB的药物浓度下，美西律的RDB开始最快，其第2个V_max所产生的抑制已占RDB的64%，奎尼丁，劳卡尼和关附甲素的RDB开始速率常数分别为每个动作电位0.165, 0.076和0.136. 美西律，奎尼丁，劳卡尼和关附甲素产生RDB的恢复时间常数分别为1.4, 9.0, 18.2和44.0 s，而且它们的恢复时间常数是不依赖于药物浓度而变化的，结果提示，关附甲素是一个慢动力学钠通道阻滞剂.     

Modelling the loss of metabolic capacities of cultured hepatocytes: application to measurement of Michaelis-Menten kinetic parameters in in vitro systems

1. The loss of metabolic capacities during culture time constitutes a major limitation for the use of hepatocyte primary cultures in in vitro metabolism measurements. A new strategy is presented that permits one to calculate the Michaelis-Menten parameters V_max and K_m from extended experiments, by modelling V_max as a variable dependent on time using exponential or sigmoidal equations. 2. This method was tested with cortisol depletion in cultured rat hepatocytes. V_max and K_m were used to calculate intrinsic clearance, and comparisons were made with methods already described in the literature. Intrinsic clearances given by our method were scaled to in vivo hepatic clearances that were close to those reported in the literature. 3. Our method could quantify the V_max decrease with culture time from estimates of time parameters, t_1/2 or t₅₀. In our system, this V_max decrease was in agreement with P450 cytochrome inactivation rates published for the rat liver. 4. In conclusion, we propose a convenient, simple and useful general method for both Michaelis-Menten parameter estimation and modelling of variations in the metabolic capacities observed in in vitro systems. Such an approach should improve the usefulness of hepatocytes in primary cultures for long-term metabolism experiments.     

Sensitivity Analysis and the Design of Gas Uptake Inhalation Studies

Abstract

Gas uptake studies analyzed by physiologically based pharmacokinetic (PBPK) models have been used to estimate metabolic parameters for many volatiles. The metabolic constants for a saturable pathway (V_max′ mg/h and K_m′, mg/L) and for a first-order process (K_f′ h^?1) are typically inferred from the decline in chemical concentration observed in closed chamber exposures. Sensitivity analysis was used to quantify the identifiability of these metabolic parameters with PBPK models for three compounds: chloroform (V_max = 2.25 mg/h), dichloromethane (V_max = 1.33 mg/h), and carbon tetrachloride (V_max=0.11 mg/h). Further sensitivity analysis related the increased ability to estimate V_max for chloroform and dichloromethane with their higher metabolic rates, indicating the value of V_max to be an important determinant in its identifiability. The optimal experimental concentration needed for estimating V_max was lower for carbon tetrachloride (12-18 ppm) than for either chloroform (740-770 ppm) or dichloromethane (680-740 ppm), and was shown to increase as a function of V_max. In addition to V_max, blood/air and fat/air partition coefficients were found to be important determinants of sensitivity to V_max estimation. Three-dimensional sensitivity surfaces were generated in order to study the combined effect of initial chamber concentration and partition coefficients on identifiability of V_max. Within the range of parameters investigated (blood/air partition was varied between 1 and 100; the fat/air partition was varied between 1 and 800), increased sensitivity toward V_max was achieved as the blood partition increased and the fat partition decreased in value. In summary, sensitivity analysis was useful in selecting appropriate initial concentrations for identifying kinetic constants and provided increased understanding of factors determining the applicability of gas uptake techniques for assessing rates of metabolism with various volatiles.     

Rat Olfactory Mucosa Displays a High Activity in Metabolizing Methyl tert-butyl Ether and Other Gasoline Ethers

Methyl tert-butyl ether (MTBE) is a widely used gasoline oxygenate.Two other ethers, ethyl tert-butyl ether (ETBE) and tert-amylmethyl ether (TAME), are also used in reformulated gasoline.Inhalation is a major route for human exposure to MTBE and othergasoline ethers. The possible adverse effects of MTBE in humansare a public concern and some of the reported symptoms attributedto MTBE exposure appear to be related to olfactory sensation.In the present study, we have demonstrated that the olfactorymucosa of the male Sprague-Dawley rat possesses the highestmicrosomal activities, among the tissues examined, in metabolizingMTBE, ETBE, and TAME. The metabolic activity of the olfactorymucosa was 46-fold higher than that of the liver in metabolizingMTBE, and 37- and 25-fold higher, respectively, in metabolizingETBE and TAME. No detectable activities were found in the microsomesprepared from the lungs, kidneys, and olfactory bulbs of thebrain. The observations that the metabolic activity was localizedexclusively in the microsomal fraction, depended on the presenceof NADPH, and was inhibitable by carbon monoxide are consistentwith our recent report on MTBE metabolism in human and mouselivers (Hong et al., 1997) and further confirm that cytochromeP450 enzymes play a critical role in the metabolism of MTBE,ETBE, and TAME. The apparent K_m and V_max values for the metabolismof MTBE, ETBE, and TAME in rat olfactory microsomes were verysimilar, ranging from 87 to 125 µM and 9.8 to 11.7 nmol/min/mgprotein, respectively. Addition of TAMIE (0.1 to 0.5 mM) intothe incubation mixture caused a concentration-dependent inhibitionof the metabolism of MTBE and ETBE. Coumarin (50 µM) inhibitedthe metabolism of these ethers by approximately 87%. Furthercomparative studies with human nasal tissues on the metabolismof these ethers are needed in order to assess the human relevanceof our present findings.     

Estimation of Octanol-Water Partition Coefficients and Correlation with Dermal Absorption for Several Polyhalogenated Aromatic Hydrocarbons

n-Octanol-Water partition coefficients (K_ow)were experimentallyestimated for: 2,3,7,8-tetrachloro- and 2,3,7,8-tetrabromodibenzo-p-dioxin;2,3,7,8-tetrachloro-, 2,3,7,8-tetrabromo-, and 1,2,7,8- tetrabromodibenzofuran;1,2,3,7,8- pentachioro-, 1,2,3,7,8-pentabromo-, 2,3,4,7,8-pentachloro-,and 2,3,4,7,8- pentabromodibenzofuran; 1,2,4,6,8,9-hexachlorodibenzofuran(HxCDF), and 3,4,3',4'-tetrachlorobiphenyl (3,4,3',4'-TCB).The method involved correlation of literature K_ow with reversephasehigh-performance liquid chromotography (RP-HPLC) retention timefor a series of 23 calibration standards and estimation of K_owfor test compounds from measured RP-HPLC retention. Retentiontimes for all standards and test chemicals were measured induplicate on the same octadecasilane (C₁₈ reverse-phase columnwith an isocratic 85:15 methanol:water mobile phase; soluteswere detected by uv absorbance (254 nm). Literature log K_owvalues used for the calibration standards had been measuredexclusively by the generator column method. Log K_ow estimatesfor the test compounds in the present study ranged from 5.45for 3,4,3',4'-TCB to 6.81 for HxCDF. K_ow estimates were thenplotted against laboratory data for the in vivo 3-day dermalabsorption of single equimolar doses (200 pmol and 20 nmol,or 1 nmol/kg and 0.1 µmol/kg) of selected test compoundsin male F344 rats. Strong inverse correlations were found betweenoctanol-water partition coefficient estimates and single-dosedermal absorption for most of the compounds studied. In addition,RP-HPLC retention time itself appeared to be as equally suitedas K_ow to such correlations with dermal absorption. The structure-activityrelationships suggested in this study were sought in order toexplain observed differences in the dermal absorption of polyhalogenateddibenzo-p-dioxin, dibenzofuran, and biphenyl congeners differingin number, position, and/or type (Cl or Br) of halogen substituents.Moreover, these results should be of predictive value in therisk assessment of dermal exposure to polychlorinated dibenzo-p-dioxins,dibenzofurans, biphenyls, and their brominated analogues.     

The Relationship between pKa and Skin Irritation for a Series of Basic Penetrants in Man

The Relationship between pK_a and Skin Irritation for a Seriesof Basic Penetrants in Man. BERNER, B., WILSON, D. R., STEFFENS,R. J., MAZZENGA, G. C., HINZ, R., GUY, R. H., AND MAIBACH, H.I. (1990). Fundam. Appl. Toxicol 15, 760–766. For a seriesof bases, which penetrate through human skin in vitro at similarrates (0.056–0.49 µM/cm²/hr), penetrant pK_ais shown to correlate with erythema, edema, and color meterreadings. As estimates of irritation, erythema, edema, and rednessmeasurements are highly linearly correlated. For the selectedseries, irritation becomes significant for bases with a pK_a> 8. The irritation potential of acids with pK_a 4 has beenpreviously reported; pK_a appears highly predictive of acuteskin irritation for acids and bases in man.     

Mechanistic Models Describing Active Renal Reabsorption and Secretion: A Simulation-Based Study

The objective of the present study was to evaluate mechanistic pharmacokinetic models describing active renal secretion and reabsorption over a range of Michaelis–Menten parameter estimates and doses. Plasma concentration and urinary excretion profiles were simulated and renal clearance (CL_r) was calculated for two pharmacokinetic models describing active renal reabsorption (R1/R2), two models describing active secretion (S1/S2), and a model containing both processes. A range of doses (1–1,000 mg/kg) was evaluated, and V _max and K _m parameter estimates were varied over a 100-fold range. Similar CL_r values were predicted for reabsorption models (R1/R2) with variations in V _max and K _m. Tubular secretion models (S1/S2) yielded similar relationships between Michaelis–Menten parameter perturbations and CL_r, but the predicted CL_r values were threefold higher for model S1. For both reabsorption and secretion models, the greatest changes in CL_r were observed with perturbations in V _max, suggesting the need for an accurate estimate of this parameter. When intrinsic clearance was substituted for Michaelis–Menten parameters, it failed to predict similar CL_r values even within the linear range. For models S1 and S2, renal secretion was predominant at low doses, whereas renal clearance was driven by fraction unbound in plasma at high doses. Simulations demonstrated the importance of Michaelis–Menten parameter estimates (especially V _max) for determining CL_r. K _m estimates can easily be obtained directly from in vitro studies. However, additional scaling of in vitro V _max estimates using in vitro/in vivo extrapolation methods are required to incorporate these parameters into pharmacokinetic models.     

Computer Modeling of the Pharmacokinetics of Fluorouracil and Thymine and Their Kinetic Interaction in Normal Dogs

The kinetic behavior of thymine and 5-fluorouracil has been shown to be non-linear and mediated largely by saturable metabolic processes. In vivo estimates of the Michaelis-Menten parameters V_max and K_m were obtained from constant infusion data in normal dogs using a system of balance equations that equate drug input with total output at steady-state. These estimates were then successfully used to simulate both steady-state and post-infusion plasma concentration-time curves for both compounds over a range of saturating and non-saturating conditions. It has been shown previously that estimates of V_max and K_m obtained from dynamic data can be incorrect if an inappropriate compartmental model is used in the analysis. Determining the Michaelis-Menten parameters at steady-state eliminates this difficulty. Moreover, the use of steady-state derived values to simulate post-infusion data confirms the validity of this technique. The kinetic interaction between thymine and 5-fluorouracil was investigated as a case of competitive metabolic inhibition in vivo by calculating K_i values from data obtained during simultaneous constant infusions of the two compounds. These values were then used in conjunction with a series of differential equations incorporating reciprocal metabolic effects to simulate the effect of thymine on FU plasma concentration.     

Adrenocortical Toxicity of 3-Methylsulfonyl-DDE in Mice: II. Mitochondrial Changes following Ecologically Relevant Doses

Transmission electron microscopy was used to characterize earlyultrastructural lesions in the adrenal zona fasciculata of femaleC57BL mice given a single ip injection of the adrenocorticolyticDDT-metabolite 3- methylsulfonyl-DDE (MCSO₂-DDE Following 3mg/kg, mitochondrial changes were observed 6 hr after dosing.At 12 and 24 hr the mitochondrial changes were conspicuous,with disorganization and disappearance of central cristae. Atdoses of 6, 12, and 25 mg/kg body wt initial (6 hr) mitochondrialvacuolization was observed, followed by disappearance of mitochondria(6–12 mg/kg) or cellular necrosis (25 mg/kg). The metabolicactivation and binding of MeSO₂-[¹⁴C]DDE in adrenal homogenateswere determined in vitro. The irreversible binding of MeSO₂-[¹⁴C]to the mitochondria-containing adrenal S-9 pellet fraction was50 times higher than that to the postmitochondrial S-12 supernatantfraction. The apparent K_m was 2.1 µM and the apparentV_max was 104 pmol/mg protein/30 mm for the binding of MeSO₂-[¹⁴C]to S-0.3 supernatants. The irreversible protein binding wasinhibited by metyrapone (K₁=1 µM) and 11-deoxycorticosterone(K₁=3 µM). In conclusion, the adrenal metabolic activationof MeSO₂-[¹⁴C]DDE is suggested to be mediated by a mitochondrialcytochrome P450 form, presumably P450 (11ß). A primarymitochondrial lesion develops and subsequently leads to degenerationand necrosis of the zona fasciculata.     

A Distributed-Parameter Model for Formaldehyde Uptake and Disposition in the Rat Nasal Lining

DNA–protein cross-links (DPX) serve as a dosimeter for inhaled formaldehyde and are associated with tumor induction in rat nasal passages after chronic exposure to 6 ppm and above. To determine the role of epithelium-specific morphometry in formaldehyde-induced patterns of injury, we developed a mathematical model that links airflow-driven formaldehyde uptake with DPX formation in regions of the rat nose with high and low tumor incidence. A three-dimensional, anatomically accurate computational fluid dynamics model of rat nasal airflow and inhaled gas uptake was integrated with a physiologically based mathematical model incorporating tissue thickness, formaldehyde diffusion, its removal by enzymatic and nonenzymatic processes, and DNA distribution in the nasal mucosa to predict DPX formation. The model implicitly incorporates the reversible conversion of formaldehyde to methylene glycol. Where possible, parameter values were taken from the literature or estimated using published correlations. The Michaelis–Menten kinetic constants V_max and K_m, as well as a first-order constant for formaldehyde removal, were left as fitted parameters. The resultant model fit to the experimentally measured DPX in the high- and low-tumor-incidence regions of the rat nasal passages was very good. Sensitivity analysis indicates that among the fitted parameters, model fits are most sensitive to V_max and that predictions were sensitive to changes in tissue thickness when all other parameters are held constant. The model structure facilitates extrapolation to primates and humans and application to other soluble, reactive gases.     

利用细菌/杆状病毒系统在昆虫细胞中表达人类UGT1A6

目的　获得单一有活性的人类UGT1A6重组酶 ,以进一步研究经该酶催化的底物谱及其在对底物作用的过程中与其他同工酶之间的相互作用。方法　利用细菌 /杆状病毒系统 ,将构建的pFsatBac UGT1A6重组质粒转化E .coliDH 10Bac大肠杆菌 ,通过转座作用获得重组粘粒Bacmid ,将其转染草地夜蛾细胞 (Sf9)后 ,产生重组杆状病毒。该病毒再感染Sf9细胞 ,即可获得人类UGT1A6重组酶。该酶的活性以 4 硝基酚为底物 ,用高效液相色谱法分析鉴定。结果　利用细菌 /杆状病毒系统 ,人类UGT1A6重组酶得到表达 ,且其对 4 硝基酚的Km 值为(0 .36± 0 .0 5 )mmol·L- 1,Vmax值为 (13.1± 1.0 )mmol·min- 1·g- 1蛋白。结论　利用细菌 /杆状病毒系统可获得对 4 硝基酚有代谢活性的人类UGT1A6重组酶 ,该酶可进一步用于其他底物的Ⅱ相代谢研究。     

Covalent Binding of Inhaled Formaldehyde to DNA in the Respiratory Tract of Rhesus Monkeys: Pharmacokinetics, Rat-to-Monkey Interspecies Scaling, and Extrapolation to Man

Covalent Bindingoflnhaled Formaldehyde to DNA in the RespiratoryTractofRhesus Monkeys: Pharmacokinetics, Rat-to-Monkey InterspeciesScaling, and Extrapolation to Man. CASANOVA, M., MORGAN, K.T., STEINHAGEN, W. H., EVERITT, J. I., POPP, J. A., AND HECK,H. D'A. (1991). Fundam. Appl Toxicol 17, 409–428. DNA-proteincross-links were formed in the respiratory tract of rhesus monkeysexposed to [¹⁴C]formaldehyde (0.7, 2, or 6 ppm; 6 hr). Concentrationsof cross-links (pmol/mg DNA) were highest in the mucosa of themiddle turbmates; lower concentrations were produced in theanterior lateral wall/septum and nasopharynx. Very low concentrationswere found in the larynx/trachea/carina and in the proximalportions of the major bronchi of some monkeys exposed to 6 ppmbut not to 0.7 ppm. No cross-links were detected in the maxillarysinuses or lung parenchyma. The pharrnacokinetics of cross-linkformation in the nose were interpreted using a model in whichthe rate of formation is proportional to the tissue concentrationof formaldehyde. The model includes both saturable and nonsaturableelimination pathways and describes regional differences in DNAbinding as having an anatomical rather than a biochemical basis.Using this model, the concentration of cross-links formed incorrespondmg tissues of different spacies can be predicted byscaling the pharmacokinetic parameter that depends on minutevolume () and quantity of nasal mucosal DNA (M_DNA). The concentration-response curve for the average rateof cross-link formation in the turbinates lateral wall, andseptum of rhesus monkeys was predicted from that of F-344 ratsexposed under similar conditions. There was significant overlapbetween predicted and fitted curves, implying that V and M_DNAare major determinants of the rate of cross-link formation inthe nasal mucosa of different species. Concentrations of cross-linksthat may be produced in the nasal mucosa of adult men were predictedbased on experimental data in rats and monkeys. The resultssuggest that formaldehyde would generate lower concentrationsof cross-links in the nasal mucosa of humans than of monkeys,and much lower concentrations in humans than in rats. The rateof formation of DNA-protein cross-links can be regarded as asurrogate for the delivered concentration of formaldehyde. Useof this surrogate should decrease the uncertainty of human cancerrisk estimates derived by interspecies extrapolation by providinga more realistic measure of the delivered concenmtion at criticaltarget sites.     

Uptake of Vinylidene Fluoride in Rats Simulated by a Physiological Model

Uptake of Vinylidene Fluoride in Rats Simulated by a PhysiologicalModel. Medinsky, M. A., Bechtold, W. E., Birnbaum, L. S., Chico,D. M., Gerlach, R. F., and Henderson, R. F. (1988). Fundam.Appl. Toxicol 11, 250–260. The purpose of this study wasto develop a physiological model to simulate the uptake of vinylidenefluoride (VDF), an important plastics monomer, in laboratoryanimals. Male Fischer 344/N rats were exposed nose-only for6 hr to concentrations of VDF ranging from 27 to 16,000 ppm.Tidal volume (mean, 1.51 ml/breath) and respiratory frequency(mean, 132 breaths/min) were not influenced by exposure concentration.Experimentally determined, steady-state blood levels of VDF,obtained by gas chromatog-raphy-head space analysis of samplesfrom rats with indwelling jugular cannulas, increased linearlywith increasing exposure concentration up to 16,000 ppm. VDFtissue/air partition coefficients were determined experimentallyto be 0.07,0.18,0.8, 1.0, and 0.29 for water, blood, liver,fat, and muscle, respectively. These values and calculated constantsfor total body elimination of VDF, K_m and V_max were incorporatedinto the physiological model. Model predictions agreed withthe experimentally determined data. Time to reach steady-stateblood levels of VDF was less than 15 min for all concentrations.After cessation of exposure, blood levels of VDF decreased to10% of steady-state levels by 1 hr. Simulation of the metabolismof VDF indicated that although blood levels of VDF increasedlinearly with increasing concentration the amount of VDF metabolizedper 6-hr exposure period approached a maximum at about 2000ppm VDF.     

根除幽门螺旋杆菌四联疗法中奥美拉唑对比格犬铋吸收的影响

目的探讨根除幽门螺旋杆菌四联疗法中奥美拉唑对比格犬体内铋吸收的影响。方法 采用双周期随机交叉试验设计,6只健康比格犬随机分为2组,分别先后po给予胶体果胶铋(B)70 mg+甲硝唑(M)250 mg+四环素(T)250 mg〔BMT〕或奥美拉唑(Ome)40 mg+BMT(70+250+250)mg(Ome+BMT),并于给药前和给药后0.5,1,2,3,4,6,8,10,12和14 h采集血样并预处理,建立火焰原子吸收光谱法并测定比格犬血浆中铋的浓度,并计算其主要药动学参数。结果 成功建立了测定铋血药浓度的火焰原子吸收光谱法,比格犬口服BMT和Ome+BMT后铋的cmax分别为21.7±5.3和(37.5±5.4)μg.L-1,AUC0-t分别为116.3±27.4和(170.1±30.4)μg.h.L-1,tmax分别为3.0±0.6和(3.0±0.6)h,(Ome+BMT)组比BMT组的cmax,AUC0-t显著升高(P<0.05)。结论 根除幽门螺旋杆菌四联疗法中奥美拉唑能显著增加比格犬体内铋的吸收。     

DNA--Protein Cross-Links (DPX) and Cell Proliferation in B6C3F1 Mice but Not Syrian Golden Hamsters Exposed to Dichloromethane: Pharmacokinetics and Risk Assessment with DPX as Dosimeter

Dichloromethane (DCM) (methylene chloride; CH₂Cl₂) is metabolizedvia a glutathione S-transferase-mediated pathway to formaldehyde(HCHO), a mutagenic compound that could play a role in the carcinogeniceffects of DCM observed in the liver and lungs of B6C3F₁, miceat 2000 and 4000 ppm. Mice but not hamsters formed DNA—proteincross-links (DPX) in the liver at DCM concentrations rangingfrom approximately 500 to 4000 ppm. The formation of DPX wasa nonlinear function of the airborne concentration of DCM. Inaddition, mice exposed to DCM (6 hr/day, 3 days) at concentrationsranging from approximately 1500 to 4000 ppm showed an increasedrate of DNA synthesis in the lung indicating cell proliferation,but increased cell turnover was not detected in mouse lung atexposure concentrations of 150 or 500 ppm. Hamsters showed noevidence of cell proliferation in the lung at any concentration,and cell proliferation was not apparent in the livers of eithermice or hamsters. An extended physiologically based pharmacokinetic(PBPK) model for DPX formation in mouse liver was developed,based on a published PBPK model for DCM (Andersen, M. E., Clewell,H. J., III, Gargas, M. L., Smith, F. A., and Reitz, R. H. (1987).Toxicol. Appl. Pharmacol. 87,185–205). The extended PBPKmodel was fitted to the DPX data using the PBPK model-estimatedarea under the curve for DCM in mouse liver as the independentvariable. Parameter estimates for HCHO disposition in the liversof mice exposed to dichloromethane were similar to previouslypublished estimates for HCHO disposition in the nasal mucosaof rats exposed to formaldehyde. Using the extended PBPK model,estimates were made of the yields of DPX presumably formed inmouse liver at the DCM concentrations used in a bioassay (Mennear,J. H., McConnell, E. E., Huff, J. E., Renne, R. A., and Giddens,E. (1988). Ann. NY Acad. Sci. 534, 343–351). The tumorincidence data in mice were fitted to the DPX yields and tothe airborne concentration of DCM as alternative measures ofexposure using the linearized multistage (LMS) model. The twodose measures yielded similar maximum likelihood estimates forthe cancer risk at concentrations from 10 to 100 ppm, but theupper 95% confidence limit on the risk was reduced by two ordersof magnitude when DPX rather than the airborne concentrationwas used as the measure of exposure. The results demonstratethat an internal dosimeter such as DPX can markedly improvethe precision of low-dose risk estimates, while having onlya minor effect on the maximum likelihood estimates calculatedwith the LMS model.     

The Steady-State Michaelis–Menten Analysis of P-Glycoprotein Mediated Transport Through a Confluent Cell Monolayer Cannot Predict the Correct Michaelis Constant <Emphasis Type="Italic">K</Emphasis><Subscript>m</Subscript>

Purpose Typically, the kinetics of membrane transport is analyzed using the steady-state Michaelis–Menten (or Eadie–Hofstee or Hanes) equations. This approach has been successful when the substrate is picked up from the aqueous phase, like a water-soluble enzyme, for which the Michaelis–Menten steady-state analysis was developed. For membrane transporters whose substrate resides in the lipid bilayer of the plasma membrane, like P-glycoprotein (P-gp), there has been no validation of the accuracy of the steady-state analysis because the elementary rate constants for transport were not known. Methods Recently, we fitted the mass action elementary kinetic rate constants of P-gp transport of three different drugs through a confluent monolayer of MDCKII-hMDR1 cells. With these elementary rate constants in hand, we use computer simulations to assess the accuracy of the steady-state Michaelis–Menten parameters. This limits the simulation to parameter ranges known to be physiologically relevant. Results Using over 2,300 different vectors of initial elementary parameters spanning the space bounded by the three drugs, which defines 2,300 “virtual substrates”, the concentrations of substrate transported were calculated and fitted to Eadie–Hofstee plots. Acceptable plots were obtained for 1,338 cases. Conclusion The fitted steady-state V_max values from the analysis correlated to within a factor of 2–3 with the values predicted from the elementary parameters. However, the fitted K_m value could be generated by a wide range of underlying “molecular” K_m values. This is because of the convolution of the drug passive permeability kinetics into the fitted K_m. This implies that K_m values measured in simpler systems, e.g., microsomes or proteoliposomes, even if accurate, would not predict the K_m values for the confluent monolayer system or, by logical extension, in vivo. Reliable in vitro–in vivo extrapolation seems to require using the elementary rate constants rather than the Michaelis–Menten steady-state parameters.