Document e7v9xQ6xq5XGwXv41RpYe4E4M

ti * K-OOW/'/eo t<lqo CAS UPTAKE INHALATION TECHNIQUES AND THE RATES OF METABOLISM OF CHLOROMETHANES, CHLOROETHANES, AND CHLOROETHYLENES IN THE RAT Michael L Gargas, Harvey J. Clewell, III Harry G. Armstrong Aerospace Medical Research Laboratory, Toxic Hazards Division, Wright-Patterson AFB, Ohio Melvin E. Andersen Chemical Industry Institute of Toxicology, Research Triangle Park, North Carolina Closed atmosphere qas uptake exposures have been used to estimate the kinetic constants o.' metabolism for the chlorinated methanes, chlorinated eihylencs. and four of the chlorinated ethanes in the male Fischer 344 rat A physiologically based pharmacokinetic (PR-PK) model was used to analyze a group of four to five uptake curves for each chemical. Eight of the fourteen compounds have been studied previ* ously by gas uptake techniques, and these data sets wem reanalyzed bv computer optimization. With 1,2-dichloroethane and methyl chloride, the PB-PK r-odet had to be expanded to account for glutathione depl^fon. while with os* and trans-7,2 dich- lornethylene the model had to be <.*panded to iccomm&Jatr swade enzyme mhibition-resynthesis to adequately describe the obsei'ed uptake behavior The ef fects of pyrazote pretreatment, known to inhibit cytochrome P-450 osidase activities, were also investigated. The kinetic behaviors determined for these compounds were either saturable, first-order, or a combination of these two processes Tetrachloroethy- fene and 1,1,1,-trichloroethane displayed behaviors consistent with first-order metabo lism only (0.3 and 5.0 respectively), indicating very low overall metabolism. The maximum metabolic rates for the chemicals exhibiting saturable reactions ranged from 2.6 ti/nol h^1 kg ~1 for carbon tetrachloride to 90 9 nmol h ~ 1 kg ' 1 for chloromethanv. The saturable reactions svere all high affinity, with K,tI values less than 20 jiM m all cases, and less than 5 jiM lor most, these kmetn constants can be utilized m PB-PK models to predict kinetic behavior for ta-mus mu:rs of exposure, varied exposure scenarios, and for interspecies extrapolation* Inwghts into structural requirements for metabolic rates arc also presented. The authors would like to than* R. Burgess. G- Cason, and I) Vmsard tor their technical contributions m conducting exposures, and K Collie* *wd (, C'l.isser for their e\p* rt assistance in the preparation of this manuscript The animals used in this study were handled in accordance with the principle* stated in the Cuide for the Ore and Use of Laboratory Animals, prepjn*d I tv tin* Commit tee on Care and Use of Laboratory Animals of the Institute ut Laboratory Animal Re sources, National Research Council, DHHS, National Institutes of Health Publication flv 2.1. 19A5, and the Animal Welfare Art of 1%h. as amended Requests for reprints should be sent to Or. Michael I. (<.ut*a\, L hemital Industry Institute of Toxicology, P.O, Box 12117, 6 Davis Drive*. Research Triangle Park NC 277O'* 295 Inhalation Toxicology, 2:295-319. 1990 Copyright 1990 bv Hemisphere Publishing Corporation 1: i 296 M. L GARCAS ET AL. INTRODUCTION Chlorinated methanes, ethanes, and ethylenes are metabolized in mammals primarily by the mixed-function oxidase system of the liver (Kubic and Anders, 1978; Salmon et al., 1981;Tvanetidvand Van Den Honert, 1981; Anders and Pohl, 1985; Henschler, 1985; Mansuy and Battioni, 1985). For all members of this group of compounds, metabolites appear to be more toxic than parent chemicals. Knowledge of the rates of metabolic activation for this group of compounds is very important for describing the disposition and elimination of the parent chemical and must be considered when evaluating expected risks associated with exposures to these compounds. Physiologically based pharmacokinetic (PB-PK) models are becom ing recognized as valuable tools in predicting the distribution of chemi cals in mammals following various routes of exposure (Ramsey and An dersen, 1984; McDougal et al., 1985; Gargas et al., 1986b; Paustenbach et al., 1988) and as a means of conducting human risk assessments (Ander sen et al., 1987a; Clewell and Andersen, 1987; Clewell et al., 1988). PB-PK models have also been used as analytical tools in deriving metabolic constants from closed-chamber exposure data (Gargas et al., 1986a). In these experiments, the PB-PK models were completely defined physio logically and as to chemical solubility in blood and tissues, lacking only the constants of chemical metabolism. Values for these kinetic con stants were incorporated into the model and varied during multiple simulations until acceptable agreement was achieved between model prediction and the observed experimental chamber concentration data (Gargas et al., 1986a), The constants that resulted in the best visual fit for the entire data set were considered optimal, although no statistical optimizations were performed. The purpose of this present work was to determine the kinetic con stants of chemical metabolism in the rat for the chlorinated methanes, chlorinated ethylenes, and four of the chlorinated ethanes by gas up take exposures coupled with analysis by a PB-PK model. The five re maining chlorinated ethanes have been examined by different methods (Gargas and Andersen, 1958b). Metabolic kinetic constants for several of these compounds have been determined previously and were used as initial values for optimization. The previously tested compounds were dichloromethane (Gargas et al., 1986b), carbon tetrachloride, 1,1dichloroethylene, 1,1,1-trichloroethane (Gargas et al., 1986a), and tri chloroethylene (Andersen et al., 1987b). Parameters of a GSH depletion model that had previously been determined for 1,2-dichloroethane (An dersen et al., 1986b; D'Souza et al., 1987) were also optimized and a similar model was developed for methyl chloride. The metabolic con stants of a previously described model incorporating suicide enzyme inhibition for the 1,2-dichloroethylenes (Andersen et al., 1986a) were i- ` to <tn0 CHLORINATED HYDROCARBON METABOLISM 297 also optimized. In addilion to methyl chloride, the chemicals for which no previous closed chamber studies were conducted were chloroform, chloroethane, 1,1-dichloroethane, vinyl chloride and tetrachloroethylene. Uptake curves for rats pretreated with pyrazole, an inhibitor of P450 metabolism (Andersen et al., 1979; Cargas et al., 1986a, 1986b), were also investigated and a preliminary qualitative analysis was performed relating molecular structure to observed metabolic rates. METHODS Animals Male Fischer 344 rats [strain designated CDF (F-334) CrIBr; Charles River Breeding Laboratories, Kingston, N.Y.] weighing betwto. 200 and 300 g were used throughout this study. The rats did not have access to food (Purina) or water during exposures. The rats were housed in a portable laminar air-flow enclosure with a 12-h on, 12-h off lighting cycle. All exposures were started between 8 and 9 a.m. Chemicals and Pyrazole Pref-eatments The chemicals studied in this work were chloromethane (CH,Cl, 99.5%; Matheson, East Rutherford, N.J.), dichloromethane (CH,CI;, 99.8%; Fischer Scientific Co.. Fair Lawn, N.J.), chloroform (CHCl,, 99.9%; Aldrich Chemical Co., Inc., Milwaukee, Wis.), carbon tetrachloride (CCI,, 99%; Matheson), chloroethane (ethyl chloride, EC, 99.7%; Mathe son), 1,1-dichloroethane (DCE, 99%; Aldrich), 1,2-dichloroethanc (ethyl ene dichloride, EDC, 99.9%; J. T, Baker Chemical Co., Phillipsburg, N.|,), 1,1,1-trichloroethane (methyl chloroform, MC, 99.8%; Dow Chemical Co., Midland, Mich.), vinyl chloride (CIV, 1.0% in purified air; Mathe son), 1,1-dichloroethylene (vinylidene chloride, VDC, 99%; Aldrich), c/'s- 1,2-dichloroethylene (C,-DC.t, 9/%; Aldrich), //ans-1,2-dichioroethylGne (T-DCE, 98%; Aldrich), trichloroethylene (TR1, 99%; Aldrich), and tetrachloroethylene (TETRA, 99%; Aldrich). The chemical purities listed above were determined by the manufacturer. The CH,CI, EC, and VCI were obtained in pressurized cylinders, with all other chemicals purchased as liquids. Pretreated animals were dosed with 320 mg pyrazole (1,2diazole)/kg, 0.5 h prior to gas uptake exposures. Pyrazole (obtained from Aldrich) was diluted ir. distilled water and administered ip in a total volume of 2.0 ml/kg. Closed Chamber Exposures The gas uptake exposure system and technique have been de scribed previously (Gargas et al., 1986a, 1986b; Gargas and Andersen, 1988a). In brief, three rats were placed in a recirculating 9.1-1 gloss chamber and were allowed to acclimate for 30 min. Oxygen was added 2<t M. L GARGAS FT AL. and maintained at -- 21% and carbon dioxide was removed with sodasorb (W. R, Grace and Co., Atlanta, Ga.). Liquid or gaseous chemical was added in a sufficient volume to achieve a targeted initial chamber con centration. The atmosphere was serially sampled with an automatic gas sampling valve every 10 min and analyzed by gas chromatography (Hewlett-Packard, Avondale, Pa.) in order to monitor the time course of chemical uptake. Four or five such exposures, lasting from 3 to 6 h, were conducted for each chemical using a range of starting concentra tions. The kinetics of the resulting uptake behaviors were very sensitive to metabolism (see Appendix). Due to chemical adsorption onto chamber surfaces, it was neces sary to determine chemical loss rates from any empty chamber. These experiments were conducted prior to animal exposures at each initial concentration utilized. For all compounds tested, the loss rates were first-order and invariant with initial concentrations. The rate constants varied between compounds, but in ail cases were less than 0.04 h'1. This loss of chamber concentration was accounted for in the mass bal ance equation for the chamber as described previously (Gargas et al,, 1986a). The chromatographic conditions used for analyzing the closed chamber atmosphere for 6 of the 14 compounds have not been previ ously presented. These compounds are CH,CI, CHCI,, EC, DCE, VCI, and TETRA. For each of these compounds (except TETRA exposures starting under 5 ppm), the gas chromatograph (Hewlett Packard, Avon dale, Pa.) was equipped with a hydrogen flame ionization detector op erated at 300C with nitrogen as the carrier gas (30 ml/min). A 10-ft, 1/8in OD stainless steel column containing 10% SE-30 on chromosorb (Supelco, Bellefonte, Pa.) was used. All analyses were conducted isotheimally at temperatures slightly above the boiling points of each compound. Under these conditions the on-column retention times were in the range of 0.7-1.5 min for these 6 chemicals. Increased sensi tivity was required for TETRA exposures starting at 1.0 and 2.0 ppm. For these exposures an electron capture detector f"Ni) was utilized. The detector was operated at 250C with 95% argon/5% methane (23 ml/min) as the carrier gas. The same column was used as described above with an isothermal oven temperature of 100C. The retention time under these conditions was 2.1 min. Data Analysis The series of uptake curves obtained for each chemical were ana lyzed using a PB-PK model originally described by Ramsey and Ander sen (1984) and modified to account for the chemical concentration in a closed atmosphere as described by Gargas et al. (1986a). The physiologi cal parameters and mass balance equations in this present formulation have been described previously (Gargas et al., 1986a; Gargas and Ander- CHLORINATED HYDROCARBON METABOI ISM 299 ;. l: sen 1988a) and the required blood and tissue solubilities for all chemi cals studied have been reported (Cargas et al., 1989). All of the parame ters in the model were thus defined except for those governing the contribution of metabolism to the decline in chemical concentration from the chamber atmosphere. Metabolism was assumed to occur ex clusively in the liver and the mass balance equation for that organ ac counted for saturable metabolism defined by V,,,,c, the maximum meta bolic rate (mg/h-kg), and Kn, the Michaelis constant (mg/I). In addition, any contributions due to first-order metabolic processes were defined with a first-order rate constant, k,c (h_1 kg"'). The model was provided with appropriate experimental parameters such as starting chamber concentrations and average body weights of the three animals, and the chamber uptake profiles were then simulated and compared to experi mental data. The kinetic constants were adjusted and simulations were repeated to obtain a satisfactory visual fit for the entire set of uptake curves using one set of constants. These values of and kl( were used as starting values in a statistical optimization software package (SIMUSOLV, trademark of the Dow Chemical Company) that varied these parameters until a best least-squares fit was achieved. The K,, was not varied during these optimizations (see Discussion). Additional equations describing glutathione depletion were re quired in the model for EDC and CH,CI (Andersen et al., 1986b; D'Souza et al., 1987). Cis- and frans-1,2-dichloroethylene required a model that incorporated suicide enzyme inhibition-resynthesis to ade quately describe the uptake data (Andersen et al., 1986a; Clewell and Andersen, 1987). The kinetic constants obtained using these models were also optimized with SIMUSOLV. Development of the equations and structure of the basic PB-PK in halation model, the models describing gas uptake, GSH depletion, and suicide enzyme inhibition can be found in recent reviews (Gargas and Andersen, 19883,-Clewell and Andersen, 1987). The sensitivities of gas uptake data to kinetic constants and several other parameters are dem onstrated in the Appendix for three different initial concentrations of dichloromethane. RESULTS Chlorinated Methanes The CHCI, gas uptake curves were adequately described by a single, saturable metabolic process. Using the optimized VmJW (Table 1) simula tions compared well with experimental data (Fig. 1). Gargas et al. (1986b1 evaluated CH,CI3 at initial closed chamber concentrations of 100, 500, 1000, and 3000 ppm. The kinetic constants obtained from these studies were consistent with both saturable and first-order metabolism (Table i TABLE 1, Kinetic Constants of Chlorinated Methanes and Tthanes Chemical Chlo romethane6 Oichloromethane Chloroform Carbon tetrachloride Chloroethane 1,1-Dichlorocthane 1,2-Dichloroethane* 1,1,1-Trichloroelhane VrfTUlC* (mg/h) 5.2C (5.53 0.001) 4.0 (3.96 0.006) 6.8 (6.81 0.004) 0.4 (0.34 0.006) 4.0 (3.95 0.002) 7.5 (7.60 0.004) 3.25 (3.93 0.006) Oimol/h) 96.9 47.1 56.9 2.6 62.0 75.8 32.8 (mgfj) 1.0 0.4 0.25 0.25 0.1 0.2 0.25 -- CkAO 19.8 4.71 2.09 1.63 1.55 2.02 2.53 -- k` <h" '> 2.0 (1.87 0.003) 1.0 (1.07 0.004) 5.0 (3.25 0.004) The constants and kfc are scaled to a 1 0-kg animal. To determine the and kt for an anirrat of any body weieht (BW In "These chemicals were analyzed using a GSH depletion model. See Olscussion for the derivation of comparable first-order rates fhese constanls were determined by visual inspection and manual adjustment. The values in parentheses were statistically optimized using SIWUSOtV. The error is the linearized standard deviation about the constant as determined by SIMUSOIV. 'I l-968MS,8d BpestW! i^iI'rnr CHLORINATED HYDROCARBON METABOLISM 30' FIGURE 1. Uptake behavior (or three rats exposed to initial CHClj concentrations of 110,500,950, 1300, and 2S50 ppm. tn this figure and all remaining figures the experimental data are represented by the symbols (x). The smooth curves were simulated using the PB-PK model containing *'atistically optimized constants (Tabic 1). These curves were best fined as a single saturable metabolic process. 1). The and k,c obtained from this earlier work were reanalyzed by computer optimization (Table 1). The fit to the curves were only slightly improved compared to earlier work. In an earlier study CC14 appeared to be metabolized solely by a low-capacity, saturable pathway (Gargas et al., 1986a). Exposures were conducted at initial chamber concentrations of 0.65,10,105, 230 ppm. The V,,.w provided by;-reanalysis (Table 1) pro vided only a slight-improvement in fit between previously reported and present results. Chlorinated Ethanes EC and DCE uptake kinetics had not been evaluated previously. EC metabolism was best described as a combination of saturable and fi'storder processes (Fig. 2). In contrast, DCE metabolism was adequately described as a saturable process only (Fig. 3). MC metabolism has been previously characterized as a first-order process (Gargas et al., 1986a). These previous uptake results obtained at 0.19, 1.1, 9.5, and 210 ppm were reanalyzed in this present study, resulting in a kle that was only slightly lower than that reported earlier (Table 1). No significant visual improvement in fit to the data was noted when using this optimized constant as compared to the fit obtained using the value of ku reported 302 M. L GARGAS ET AL FIGURE 2 Chloroethane (CE) uptake curves for three rats starting at 100, S3S, 1200, and 2350 ppm. The smooth curves were simulated using statistically optimized constants (Table 1) indicative of both saturable and first-order processes. FIGURE 3. 1,1-Dichlorcx''.hane (DCE) metabolism was characterized by a single saturable process. These uptake curves at 90, 490, 1100, and 2175 ppm were fitted using statistically optimized con stants (Table 1). CHLORINATED HYDROCARBON METABOLISM 303 in the earlier work. Either of these values (Table 1) indicates only a low rate of metabolism for MC. Chlorinated Ethylenes VCI and TETRA are the two ethylenic chemicals that had not been previously studied by gas uptake techniques in our laboratory. VCI me tabolism was satisfactorily described by a combination of saturable and first-order components (Fig. 4 and Table 2). TETRA metabolism was best modeled as a slow first-order process. Optimization produced a k,r much smaller than estimated visually, and the estimated standard devia tion was larger than for other chemicals (Table 2). At this low rate of metabolism, only slight differences were observed in the apparent qual ity of the fits obtained when using either of these rate constants (Fig. 5, only optimized results are presented). Cargas et al. (1986a) exposed rats to initial concentrations of VDC at 210, 550, 2730, and 10,000 ppm, and Andersen et al. (1987b) evaluated TRI at 100, 480, 1000, 2000, and 4640 ppm. These earlier studies demon strated uptake behavior consistent with saturable metabolism for both compounds. Reanalysis gave V'w values that compared very well with constants previously obtained (Table 2). EDC and CH,CI/GSH Depletion Model Earlier attempts at fitting uptake data for EDC required metabolism characterized by a saturable process as well as a variable first-order process assumed to involve CSH conjugation (Andersen et al,, 1986b; D'Souza et al., 1987). To obtain adequate fits it was necessary to de crease the klc as the exposure concentrations increased, consistent with previous observations of a progressive depletion of GSH. A GSH deple tion model was devised (Andersen et al., 1986b; D'Souza et al., 1987) that included a second-order rate constant describing the conjugation between GSH and parent chemical, Kt, (0.0014 h'1 The values describing the saturable oxidative EDC metabolism were a of 3.25 mg h_1 kg-' and Km of 0.25 mg/I. The GSH parameter values have been refined by D'Souza et al. (1987) and were used in this present study. For our purposes, the GSH parameters were held constant and was varied. The value of (Table 1) was found to be only moderately increased by optimization as compared to the previously reported results. Preliminary examination of uptake data for CH,CI was consistent with GSH depletion similar to that observed with EDC. The GSH deple tion model (D'Souza et al., 1987) was therefore used to analyze the CHjCI uptake curves. Initial visual estimates were made for (5.2 mg kg-1 h_1), Km (1.0 mg/I), and K(0.01 h-1 the second-order rate constant for CH,CI conjugation with GSH. In order to validate these values were used to simulate GSH depletion experiments during R&S148965 CHLORINATED HYDROCARBON METABOLISM 4 305 FICURE 4. Vinyl chloride uptake at 23S, 573,1250, 3ISO ppm starting concentrations. The smooth curves were generated using the kinetic constants obtained statistically (Table 2). VCI metabolism was characterized by both saturable and first-order processes. constant-concentration inhalation exposure to CH3CI at 500,1500, 2000, and 2500 ppm for 6 h (data from Kornbrust and Bus, 1984). Hepatic GSH levels measured at the end of the 6-h exposures were expressed in terms of percent control GSH. These results and those predicted by the GSH depletion model described here were compared (Table 3). The second-order conjugation of GSH with CH3C! (Kconsistent with simu lations was 0.009 h~" pM~\ The gas uptake curves were then evaluated with all GSH parameters fixed, allowing only to be varied (Table 1). A comparison of simulated and experimental gas uptake results using these constants was excellent (Fig. 6). T-DCE and C-CDE/Suicide Inhibition-Resynthesis Model Previous attempts at fitting gas uptake data for T-DCE and C-DCE using the basic gas uptake model provided results suggestive of de creasing metabolic activity during the course of exposure (Fig. 7). An dersen et al. (1986a) explored four possible mechanisms for enzyme inhibition. The only description that provided adequate simulation of the uptake kinetics assumed that short-Mved reactive metabolites pro duced during 1,2-dichloroethylene metabolism interacted with the enzyme-1,2-dichlorethylene complex, resulting in enzyme inhibition. A suicide inhibition-resynthesis model was developed (Andersen et al., 1986a; Clewell and Andersen, 1987; Gargas et al., 1988) and kinetic con- $ 306 M. L GARCAS FT AL FIGURE S, Tetrachloroethylene metabolism was modeled using a very slow first-order constant (Table 2). Initial concentrations were 1.0, 2.1,19, 90, and 1020 ppm. stants were estimated including a second-order rate constant, Kd, de scribing enzyme inhibition, and a rate accounting for enzyme resynthe sis. This model adequately described the observed uptake kinetics. For this present work, the values of and Kd determined by An dersen et ai. (1986a) for T-DCE and C-DCE were optimized (Table 2). These optimized constants utilized in the enzyme inhibition model sim ulated the observed uptake kinetics for T-DCE and C-DCE very well (Figs. 8 and 9, respectively). TABU 3. CHjCI CSH Depletion 6-h Exposure concentration (ppm) Percent of control experimental' Percent of control simulated6 SOO 1500 2000 2500 80 28 18 15 55 30 25 20 'Percent CSH estimated from Fig. 1 of Kornbrust and Bus (19S4). ^Simulations conducted using the GSH depletion model (Andersen et al., 1986b; D'Souza ct al., 1987). CHLORINATED HYDROCARBON METABOLISM 307 FIGURE 6. Uptake behavior of CHjCI at starting concentrations of 106, 510, 2000, and 5240 ppm. The smooth curves were obtained using a PB-PK model containing equations accounting for GSH depletion. The behavior was consistent with metabolism consisting of both a saturable process (Table 1) and a concentration-dependent GSH conjugation reaction {K^ - 0.009 h"1 ^M-1) with depletion. Pyrazole Pretreatments Animals pretreated with pyrazole were exposed to an initial concern tration of 600 ppm EC (Fig. 10). Comparison of the pyrazole-treated ani mals with naive (non-pretreated) animals exposed at approximately the same starting concentration reveal distinct changes in uptake kinetics. The smooth curve (Fig. 10) was produced by the PB-PK model with V set to zero, indicating microsomal oxidation was substantially, if not completely, inhibited. Similar results were obtained in earlier work with CH2Ci2 (Gargas et al., 1986b), CCI4, MC, and VDC (Gargas et al,, 1986a). In this present work, all the chemicals were investigated with pyrazoletreated animals and in all instances exhibited behavior similar to EC (Fig. 10), with the exception of CH3CI. Pvrazole-pretreated animals ex posed to an initial concentration of 2000 ppm CH,CI produced uptake behavior only slightly different from correspondening naive animals (Fig. 11). Computer prediction using the GSH depletion model with set to zero did not adequately describe the experimental data for pyrazole pretreatment with CH3CI exposure. 308 M. L GARCAS ET AL FIGURE 7. Uptake behavior of T*DCE at initial concentrations of S, 7.2S, 10.5, 25, and 1125 ppm. The simulated curves were the best that could be produced using the PB-PK gas uptake model assuming only saturable metabolism - 0.28 mg kg~`h":. '8. Same data set ter T-DCE as in Fig i he smooth curse was cenet4U*d using the su*o<ie inhibition-is. I', model and the oDtirrvrr*d tonstarn, m Tahir* 2 CHLORINATED HYDROCARBON METABOLISM 309 T: FIGURE 9. Uptake behavior of C-DCE at initial concentrations of 70,155, 210, 950, and 2050 ppm. The behavior was adequately simulated using the suicide inhibition-resynthesis model using opti mized constants (Table 2). Q-n n n n n.a oonnonrvni-iofionn PYRAZOLE 101- f NAIVE 0.0 1.5 3.0 4.5 6.0 T (HRS) FIGURE 10. Comparison of the uptake behavior of pyrazole-pretreated and naive animals at ap proximately 600 ppm EC. The smooth curve was produced using the PB-PK model with set to WRtraraJ R&S148970 310 M. L. GARGA5 FT AL FIGURE 11. Uptake behavior of pyrazolc-treated and naive animals at 2000 ppm CHjCI. The smooth curve was predicted using the PB-PK CSH depletion model with V,^, set to zero and al! other parameters held constant. DISCUSSION This work included a statistical optimization for determining best-fit values for metabolic constants based on a least-squares analysis of gas uptake data for eight previously tested compounds and six additional compounds. The statistical approach provided linearized standard devi ations about t;-e constants. These confidence intervals convey only a rough indication of the uncertainty in the parameter estimate. In gen eral, they tend to underestimate the variability of a given parameter because they are calculated only in the local vicinity of the best-fit pa rameter value with all other model parameters held constant. Effects of correlation with other parameters and the global behavior of the model are ignored. The linearized standard deviations, however, are of some use for relative comparisons of parameter identifiability with different applications of the same model. The optimization program does finetune the visual results and provides a statistical estimate of the range for the resulting optimized parameters. Computerized optimization is a powerful tool and can greatly re duce the work of arriving at a good description of the series of curves. However, it would be unwise if it were used to replace the iterative process where the investigator adjusts kinetic values and observes CHLORINATED HYDROCARBON METABOLISM 3t1 - changes in predicted behavior (see Appendix). The insights gained by performing the manual adjustment, repeated simulation, and visual in spections are extremely valuable to the investigator for understanding the influence of the various parameters on the uptake behavior. These insights are often lost when sole reliance of choosing the appropriate constants is placed on a computer. Moreover, the performance of any numerical optimization algorithm is dependent on the initial estimate of the parameters to be varied. The algorithm may provide a parameter estimate that is only a local, as opposed to global, optimum. This prob lem is particularly troublesome for nonlinear models containing a large number of multiply correlated parameters, as is the case for most PB-PK models. Application of a numerical optimization algorithm for these models requires an interactive approach in which the investigator mon itors the performance of the optimization and actively participates in the parameter estimation process. Because and Km were highly correlated and could not be inde pendently estimated, Km was not varied during these optimizations. Values of Km above those reported, however, do not allow acceptable fits to the data no matter how Vmuc is varied. This provided evidence that the values of Km reported here are an upper limit for this parame ter. TETRA and MC are examples of poorly metabolized solvents in which gas uptake technques are not sufficiently sensitive to adequately define the slow kinetic behavior (Tables 1 and 2). Gas uptake methods are also insensitive when applied to compounds that are readily metab olized but have relatively high blood and tissue solubilities, as was dis covered with the higher substituted chlorinated ethanes (Gargas and Andersen, 1988b). For TETRA and MC, an alternative in vivo technique to gas uptake would be to estimate rates of metabolism based on me tabolite accumulation following inhalation exposures at constant con centrations. For example, MC is known to be metabolized to 2,2,2- trichloroethanol using rat liver microsomes (Ivanetich and Van Den Honert, 1981), and TETRA metabolism yields trichloroacetic acid using rat hepatocytes (Costa and Ivanetich, 1984). The gas uptake approach does have its limitations but is still a valuable tool for studying the metabolic characteristics of a variety of inhaled compounds. The relative ability of EDC and CH,C! to deplete GSH levels can be estimated by comparing their effective first-order rate constants to the fixed k,c values determined for other compounds. The effective first- order rates or (k,) for these compounds are the product of the second- order rate, and the basal GSH concentration before depletion (7000 fiM; D'Souza et al., 1987). For EDC this rate is 9.8 h"1 (he., 0.0014 h'' fiM~' x 7000 fiM) and for CH^I it is 63 h"' (i.e., 0.009 h'' M~' x 7000 ixM) in a 250-g rat. These values can be scaled to a 1.0-kg animal using the relationship k, -- kw x BW~' (bottom of Table 1), resulting in com 312 M. L. GARGAS ft AL parable k,c values for EDC and CH-,CI of 6.5 and 41.6 h"1, respectively. This pseudo-first-order rate for EDC is higher than any of the k,t values oh other compounds in Tables 1 and 2 and the constant for CH,CI is larger yet, indicating the potential of these two compounds to deplete hepatic GSH levels. In our analysis, CH3CI metabolism was characterized by a saturable component and a GSH-consuming component. The failure of pyrazole pretreatment to inhibit the saturable CH3CI process suggests that P-450- catalyzed oxidation is not the major pathway for metabolism of this chemical. Pyrazole had no apparent effect on the GSH-dependent path way. At this time, the enzymic or metabolic pathway responsible for the saturable reaction of CH,CI cannot be elucidated. The fits achieved between simulated and experimental results are constrained by the physiological description of the animals. When sim ulations do not adequately predict experiment, such as with T-DCE and C-DCE, the investigator is forced to examine the biological basis of the model failure. Filser and Bolt (1979) examined the kinetics of T-DCE and C-DCE using gas uptake techniques and a two-compartment descrip tion for analysis. The compartmental approach used by Filser and Bolt (1979) was not physiologically constrained and, although their compart ment fitting routine very adequately described their data, suicide inhi bition was overlooked. They obtained values of 7 and 25 /imol h"' kg-1 for T-DCE and C-DCE, respectively, as compared to values of 30.9 ^mol h'1 kg'1 obtained for each of the two compounds when enzyme inhibition was considered. Similar values might be expected for these two compounds considering their chemical structures. The for T-DCE obtained by Filser and Bolt (1979) seems low for the series of chlorinated ethylenes and most probably reflects an influence of en zyme inactivation in these studies. Our discovery of suicide inhibition [consistent with the work of Costa and Ivanetich (1982)] and the GSH depletion process described here and in previous work were a direct result of analyzing data within a physiologically realistic framework. Molecular modeling approaches have been used with only limited success in a quantitative structure-activity relationship (QSAR) study for describing the differences in metabolic rates observed with the chlorin ated methanes, ethanes, and ethylenes (Gargas et a!., 1988). However, a more qualitative approach utilizing the V,mi,c values in Table 1 and from Gargas and Andersen (1988b) does provide some insights into meta bolic rates. Dichloromethane, chloroform, and carbon tetrachloride can be thought of as having structural analogs in the ethane series where a substituent, R, represents one of the cnlorine atoms for the methanes, while in the ethane series, R could be -CH,, -CH.CI, -CHCh, or -CC1; (Table 4). A relative ranking of the metabolic activity found in the meth ane series is CHCI3>CH3CIj>CCI,,. A less than 10 fimol/h can be considered a low rate of metabolism, 10-50 /xmol/h a moderate rate. CHLORINATED HYDROCARBON METABOLISM 313 TABLE 4. Chlorinated Methane and Ethane Structural/Metabolic Analogs General structure H / Cl-C-R 1 H Cl / Cl-C-R / H Methane R ^nuK (Amol/h) -Cl 47.1 -Cl 58.6 R -CH, -- CHjCI -CCI, -CH} -CH2CI -CHClj Ethane (t-mol/hr 62.0 31.8 38.7* 75.8 57.7* 71 .S' -CCI, 45.5' Cl / Cl-C-R / -Cl 2.6 -CH, 0.0 Cl -CCI, 8.4J Values from Cargas and Andersen (1988b). and >50 //mol/h a high rate. The structural analogs in the ethane series show somewhat the same rankmg as their methane series counterparts with only two exceptions (Table 4). One analog of CH2Cl2, chloroethane, has a rate that would seem more appropriate for the CHCI, group, and one CHClj analog, pentachloroethane, seems to better correspond to rates found in the CH2Cl2 analog series. The compound 1,1,2trichloroethane could have been placed in either the CH,CI2 or CHCI, analog series, but based on metabolite studies carried out by lvanetich and Van Den Honert (1981), it appears 1,1,2-trichloroethane should be an analog of CHCI, in this scheme. They found monochloroacetate as the major metabolite of 1,1,2-trichloroethane and proposed a mecha nism for the pathway with oxidative attack primarily on the carbon with two chlorine substituents. This places the compound in the CHCI3 ana log series in our scheme. Several statements can be made based on the relative rankings found here. A hydrogen must be present on the carbon in the general structure (Table 4) for moderate to high rates of metabolism to occur. Carbon tetrachloride and the two ethane analogs, 1,1,1-trichloroethane and hexachloroethane, do not meet this criterion and exhibit very low rates of metabolism. Further, the rate progresses from moderate to high if two chlorines and a hydrogen are present on the carbon in the gen eral structure, except with pentachloroethane. Steric constraints may 314 M. L CARCAS FT AL be important in the case of pentachloroethane due to the presence of three chlorines on the carbon substituent, and this might explain the observed moderate rate of metabolism as opposed to the higher rate expected within this scheme. No explanation is obvious for the higher rate observed with chloroethane. CH,CI is not included in this analysis because it has qualitatively different metabolic characteristics (Fig. 10). In the ethylene series (Table 2), the highest metabolic rates were found in molecules with two chlorines on one carbon and at least one hydrogen on the other (trichloroethylene and 1,1-dichloroethylene). Molecules having no more than one chlorine on either carbon (vinyl chloride and c/s- and trans-1,2-dichloroethylene) had moderate rates of metabolism, and when no hydrogens were present (tetrachloroethylene) a very low rate was observed. This is consistent with the observa tions made on the methane and ethane series of chlorinated com pounds described above. As was suggested following QSAR analyses for (Gargas et al., 1988), specific electronic and steric information will have to be included for successful development of a predictive model. This work provides kinetic constants of chemical metabolism for the industrially and environmentally important chlorinated methane, ethane, and ethylene series of compounds. The statistical optimizations increased confidence in the constants obtained in previous work, and this approach was also successful in determining kinetic constants for an additional six compounds not studied previously. These constants can be used in other PB-PK models for these chemi cals in which alterations in dose route, exposure scenario, exposure concentration, or species extrapolation are used. Constants obtained by earlier gas uptake studies have already been utilized in this manner. CHjCIj constants have been used in a human risk assessment (Ander sen et al., 1987a), CCI4 constants have been used to model unusual exposure scenarios (Paustenbach et al., 1988), EDC constants have been used to simulate blood levels of EDC following oral dosing (D'Souza et al., 1987), results for several dihalomethanes have been used in a PB-PK skin penetration model (McDougal et al., 1985, 1990), and those for VDC were shown to give consistent predictions for a diverse data base for disposition (D'Souza and Andersen, 1988). The results from VDC and TRI have been used in describing a competitive inhibition model for atmospheres containing both compounds simultaneously (Ander sen et al., 1987b). The constants provided here should aid other investi gators in future endeavors for analyzing the results of a wide variety of toxicological studies with these halogenated chemicals. We would sug gest however, that for poorly metabolized substrates such as TETRA and MC, alternate in vivo techniques for assessing metabolism will be re quired before compete PB-PK models can be developed. R&S148975 CHLORINATED HYDROCARBON METABOLISM 7'5 APPENDIX Computer simulation techniques can be used to determine kinetic constants of chemical metabolism from gas uptake data because of the differential influence of various metabolic constants at different cham ber concentrations. Dichloromethane (CH^CIi) metabolism is character ized by both saturable and first-order kinetic processes with a of 4.0 mg/kg-h, Km of 0.4 mg/I, and k<c of 2.0 h-' (Table 1). Figures 12-14 present examples of the effects of various physiological and metabolic parameters on CH3C12 disappearance from the chamber at relatively low, intermediate, and high chamber chemical concentrations. At low chamber concentrations (less than 200 ppm) microsomal metabolism is first-order and perfusion limited (Fig. 12). The declining chamber con centration is driven by the metabolic constants V^ and Km. Because and K,, are highly correlated, they are not independently identifi able from gas uptake studies alone. Previous studies using metabolite measurement (Gargas et al., 1986b) and mixed exposures (Andersen et al., 1987b) showed that P-450 metabolism of these chlorocarbons is, in general, a high-affinity process with a Km well below 1.0 mg/I. Alveolar FIGURE 12. Scnsitiv ty of CH2CI2 gas uptake data to metabolic and physiologic parameters a: an initial chamber concentration of 100 ppm. The best-fit simulations were produced usinp the mid die values listed for each parameter. For each panel, the best-fit parameters were n\od and onl\ the listed parameter was varied as noted. R&S148976 316 M. L CARGAS ET AL FIGURE 13. Sensitivity of CHjCIj uptake data to variou-- parameters at an initial concentration of 500 ppm. ventilation (Q^) also significantly affects the rate of uptake at these lower concentrations. Another physiological parameter, liver blood flow (Qj, is a limiting factor that influences the first-order decline, but to a lesser degree than ventilation (Fig. 12c). The rate constant k,c has very little effect at lower concentrations even when varied from 0.0 to 4.0 h_1 (not shown). At intermediate chamber concentrations of 500-1000 ppm (Fig. 13), the uptake behavior becomes sensitive to both increasing and decreas ing Vmlic. The Km becomes important only as chamber concentration falls during the course of the exposure (Fig. 13b). The chamber concen trations at which the transition from saturable to first-order metabolic clearance occurs are dependent on VMK and, to a lesser extent, /Cm. First-order metabolic contributions from klr begin to have an influence at intermediate concentrations and become more prominent as cham ber concentrations a~e increased above saturating concentrations (Fig. 14b). Alveolar ventilation (Q^) has an influence on the behavior at inter mediate concentrations (Fig. 13d) but is less influential at the highest chamber concentrations (Fig. 14d). Only the early uptake behavior at 3000 ppm is affected by Q,* (Fig. 14d). First-order metabolic contribu tions (kk) influence the higher concentration uptake behaviors the Fl&S148977 CHLORINATED HYDROCARBON METABOLISM 377 most, with less important as compared to intermediate concentra tions. The K,, has very little impact on the chamber decline at high chamber concentrations. Blood solubility alters the initial extent of up take, but has little influence on the shape of the later time points (Fig. 14c, notice the three simulations are parallel after approximately 2.0 h). For the manual fitting of kinetic constants, tissue solubilities deter mined experimentally are fixed in the model as are all physiological parameters (i.e., Qx, Qk, etc.). The and Km are initially adjusted to fit the data at intermediate initial concentrations (Fig. 13), with addi tional adjustments performed at iower concentrations if necessary (Fig. 12). The value of kh is set to zero for these initial simulations. The best V,,uc and Km are then tested with higher concentration data to see if first-order metabolic processes are present. First-order metabolism is suspected if the V,miIC and Km used to fit the intermediate and lower concentration curves consistently underestimate the decline in experi mental data obtained at high * Micentrations. If this occurs, kic is in creased to fit these high-concentration uptake curves. The lower and intermediate curves are then reevaluated using all three kinetic con stants with minor adjustments made until the entire data sef (usually four to five curves) is adequately fitted with one set of constants. In the (PPM) FIGURE 14, Sensitivity of CH2GU uptake data to various parameters at .in initial concentration of 3000 ppm. 31 M. t. GARGAS ET AL case of chemicals with only saturable metabolism, the higher concen tration curves are generally well described using the ^ and Km ob tained from evaluating the lower concentration curves (such as with CHCIj, Fig. 1). The uptake behavior with chemicals having only firstorder metabolism (i.e., no saturable component) is characterized by a series of uptake curves that demonstrate uniform decline in chamber concentration (i.e., parallel lines at all concentrations, such as observed with tetrachloroethylene in Fig. 5). REFERENCES Anders, M. W., and Pohl, L. R. 1985. Halogenated alkanes. In Bioactivation of Foreign Compounds, ed. 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