Chemical Industry Institute of Toxicology, 6 Davis Drive, PO Box 12137, Research Triangle Park, North Carolina 27709 * Visiting scientist from the Toxicology Division, Wright-Patterson AFB, Ohio.
Received May 17, 1999; accepted September 21, 1999
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ABSTRACT |
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Key Words: inhaled methanol; metabolism; physiologically based pharmacokinetic model; relative respiratory uptake (RRU); lung-only exposure system..
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INTRODUCTION |
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Methanol is primarily cleared from systemic circulation by metabolism and to a lesser degree by exhalation. Methanol is metabolized in non-human primates by alcohol dehydrogenase to formaldehyde (McMartin et al., 1975), which is highly reactive and not thought to be directly involved in the manifestations of methanol toxicosis (McMartin et al., 1979
). Formaldehyde is rapidly converted by formaldehyde dehydrogenase to formic acid, which, under physiological conditions of the body, dissociates to formate and hydrogen ions, which cause the metabolic acidosis (Tephly and McMartin, 1984
). Formate is converted by a folate-dependent multi-step pathway to carbon dioxide (Eells et al., 1983
; Johlin et al., 1987
).
Both methanol and formate are endogenous chemicals with body burdens arising from dietary sources and metabolic processes. Methanol is available in the diet from the ingestion of fruits, fruit juices, alcoholic beverages, and certain vegetables, as well as from the gastrointestinal hydrolysis of the artificial sweetener, aspartame (10% methanol by weight; Stegink et al., 1989). Methanol is generated metabolically by the action of a methyltransferase system, although other enzymatic pathways likely contribute to endogenous levels. The endogenous methanol blood concentrations for humans range from 0.01 to 0.11 mM for methanol (Lee et al., 1992
; Sedivec et al., 1981
). Similar endogenous blood formate concentrations (0.10.2 mM) have also been reported for monkeys (Horton et al., 1992
; McMartin et al., 1979
) and humans (Buttery and Chamberlain, 1988
).
One of the interesting properties of methanol is that only a fraction of the inhaled methanol vapor is absorbed across the respiratory tract into the systemic blood circulation. In early work, using an in vitro rabbit trachea preparation, retention of lipophilic chemical vapors was compared to water-soluble chemical vapors (Fiserova- Bergerova, 1983). The percentages of retained in-flowing vapors varied among 2.5, 6.7, 8.3, and 10.6 for methylene chloride, halothane, trichloroethylene, and toluene to 48.2, 54.0, 58.2, and 68.6 for 1-butanol, 1-propanol, ethanol, and methanol. These results demonstrated that water-soluble vapors are reversibly retained in the respiratory airways.
This phenomenon is referred to as the "wash-inwash-out" effect, where during inhalation, a polar solvent is adsorbed by or dissolves into the respiratory airway and lung mucus (wash-in) and during exhalation the polar solvent desorbs (wash-out) from the respiratory airway and lung mucus and is exhaled (Johanson, 1991). The net effect is that less inhaled methanol is transferred into the lung blood supply than is anticipated, based on its blood:air partition coefficient value.
Johanson (1991) proposed a multi-compartmental model of the lung to describe fractional respiratory uptake of water-soluble vapors such as alcohols. Perkins et al. (1995) estimated relative respiratory uptake (RRU)-factor values for inhaled methanol in rodents, monkeys, and humans using a one-compartment model. Systemic clearance of methanol by metabolism was described by Michaelis-Menten kinetics. The calculated RRU-factor values for humans ranged from 0.65 to 0.79 and from 0.70 to 0.86 for rodents. That is, in humans, between 65 to 79% of inhaled methanol was absorbed into systemic circulation, and in rodents, between 70 to 86%. The methanol retained in the respiratory mucus during inhalation of methanol vapors (1-RRU) was exhaled. Perkins et al. (1995) estimated an RRU-factor value of 0.69 for male rhesus monkeys. Recently, Pastino et al. (1997) estimated RRU-factor values for inhaled ethanol in humans and rodents using a physiologically based pharmacokinetic (PBPK) model. These authors report RRU-factor values for ethanol of 0.62 for humans and 0.60 to 0.65 for mice and rats.
The objective of this research was to quantify the relative respiratory uptake of methanol in the lungs of female Cynomolgus monkeys during 2-h inhalation exposures of methanol vapors ranging from 10 to 900 ppm, using methanol breath data from the Dorman et al., 1994 study. Unreported time-course data were evaluated for inhaled and exhaled [14C]-methanol concentrations measured during [14C]-methanol vapor exposure in female Cynomolgus monkeys. Horton et al. (1992) published a PBPK model for inhaled methanol in rats, mice and monkeys. However, the authors did not account for fractional uptake of inhaled methanol.
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MATERIALS AND METHODS |
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Determination of RRUL-factor Values
A 4-compartment PBPK model (Ramsey and Andersen, 1984) was modified to account for fractional respiratory uptake of inhaled methanol (Fig. 1
) in the lung (RRUL). Fractional uptake of inhaled methanol vapors was accounted for in a similar manner as inhaled ethanol vapors in rodents and humans (Pastino et al., 1997
). Systemic intake of inhaled methanol vapors in the lung was expressed as a fraction of the inhaled methanol concentration (CI, Equation 3) and was referred to as the relative respiratory uptake (RRUL) factor. PBPK model parameter values for fractional blood flow values (Table 1
) were taken from Forsyth et al. (1968, 1971) and fractional volume of compartments (Table 1
) from Crank and Vinegar (1992). Methanol partition coefficient (PC) values reported for the human (Fiserova-Bergerova and Diaz, 1986
) were used for the PBPK monkey model (Table 1
).
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For each monkey, simulations were conducted using the calculated average ventilation rate over the 2-h [14C]-methanol-vapor exposure period (Table 2). Cardiac output was assumed to equal the ventilation rate. After exposure, cardiac output and ventilation rate were assumed to return to the normal resting conditions of 100 l/h by 4 h after cessation of anesthesia. Ventilation rate and cardiac output values, both during and after [14C]-methanol-vapor exposure, were incorporated into the PBPK model with the table function in ACSL 11 software (Mitchell and Gauthier Associates, Inc., Concord, MA). In addition, the average measured [14C]-methanol-vapor exposure concentration and the body weight of the monkey at the time of exposure were used for each monkey-exposure simulation.
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RRUL-factor values were estimated by optimization for each of the 4 targeted exposure concentrations (10, 45, 200, and 900 ppm) per monkey. RRUL-factor values were obtained by optimizing the value of RRUL to predict the amount of [14C]-methanol exhaled (AX, mg) over the 2-h exposure period. Body weight, average [14C]-methanol-exposure concentration, and calculated average ventilation rate during the 2-h exposure were used for each optimization (Table 2). Optimizations were conducted with an initial RRUL value of 0.57 and Vmaxc and Km values were set at optimized values of 15.54 and 0.66 mg/l. Other model parameters are given in Tables 1 and 2
.
Model Equations
The rate of metabolism of methanol to formaldehyde in the liver (RAM, mg/h) is described as,
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where Vmax (mg/h) is an allometrically determined metabolic rate constant (Vmax = Vmaxc*BW0.75) and Cvl (mg/l) is the venous blood concentration of methanol leaving the liver.
Systemic uptake of methanol was mathematically described by assuming that only a fraction (RRUL) of the inhaled methanol concentration was available in the alveolar air to cross the lung into systemic circulation, due to absorption of methanol into the mucus layer of the lung. The amount of inhaled methanol (AI, mg) available for uptake into systemic circulation is described as,
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where CI is the measured average inhaled methanol concentration (mg/l) and QP is the average measured ventilation rate (l/h). The quantity of methanol remaining in the mucus (1 RRUL) was desorbed by exhalation. The amount of exhaled methanol (AX in mg) is described as,
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where the first integration accounts for exhaled alveolar methanol and the second integration accounts for desorbed methanol from the respiratory tract. Ca is arterial blood methanol concentration (mg/l) and Pb is the blood/air partition coefficient for methanol.
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RESULTS |
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The amount of exhaled [14C]-methanol collected at the end of exposure increased in a linear fashion, with the exposure concentration (Figs. 2a2d) for each monkey as indicated by linear regression R2 values of 0.97 to 0.99. For Monkey 3, the 200-ppm exposure resulted in apparently erroneous data and was not included in the linear regression analysis (Fig. 2c
).
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Figures 3a3d depict the measured mean blood [14C]-methanol concentrations (±SD) during exposure and one-h postexposure and computer-simulated blood concentrations using single values for RRUL (0.59), BW (4.46 Kg), and QC and QP (34.5 l/h) obtained by averaging across exposure groups. Despite the variability in the measured blood concentrations for each exposure group, the computer-predicted mean [14C]-methanol blood concentrations were within a factor of 2 of measured values during uptake and clearance of [14C]-methanol in the blood, with the exception of the 45-ppm exposure group. Uptake of [14C]-methanol was generally under-predicted in the 10- and 45-ppm exposure groups (Figs. 4a and 4b
) and systemic clearance of [14C]-methanol tended to be slightly over-predicted at one-h postexposure across exposure groups.
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DISCUSSION |
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Johanson (1991) reported an RRU-factor value of 0.58 for methanol in humans using data from Sedivec et al. (1981). In contrast, with human methanol, pharmacokinetic data from Leaf and Zatman (1952), Lee et al. (1992), Sedivec et al. (1981),and Perkins et al. (1995) estimated RRU-factor values of 0.65 to 0.79 under resting conditions and an RRU-factor value of 0.50 during exercise. Perkins et al. (1995) also reported RRU-factor values for rats and mice ranging from 0.70 to 0.88 and suggested that the value of RRU may be dependent on the methanol-exposure concentration. In monkeys, Perkins et al. (1995) reported an RRU-factor value of 0.65 for male rhesus monkeys exposed to 1200 and 200 ppm methanol using data from Horton et al. (1992). In close agreement with Perkins et al. (1995), we report average RRUL-factor values of 0.56 and 0.61 for 200- and 900-ppm methanol lung-only exposures in female Cynomolgus monkeys.
RRU-factor values are also reported for ethanol. An RRU value of 0.55 was reported for ethanol vapors in humans (Kruhoffer, 1993). More recently, Pastino reported RRU-factor values of 0.62 for humans and 0.60 to 0.65 for mice and rats.
This PBPK model demonstrated the need to account for the "wash-inwash-out" phenomenon with inhaled methanol, even with a lung-only exposure system. Retention of methanol might have been more pronounced if the upper respiratory tract was not bypassed with an endotracheal tube. Fractional uptake of methanol was evaluated as a function of exposure concentration of methanol (10900 ppm) and exposure duration (0.5 to 2 h), using unpublished data from the Dorman et al., 1994 study. The "wash-inwash-out" phenomenon for methanol in the lung behaves as a simple linear process over a wide range of exposure concentrations and for various exposure durations, up to 2 h. This is an important observation because this kinetic analysis supports the contention that respiratory uptake of polar solvent vapors can be simulated using PBPK models by simply adjusting the inhaled exposure concentration and measuring or estimating the breathing rate. This approach will be adequate if linearity in the "wash-inwash out" phenomenon is maintained.
In this model, predictions of the amount of methanol in exhaled breath (AX) were very insensitive to Vmaxc and Km values for the metabolism of methanol. For example, a 10-fold reduction in Vmaxc or Km values or a 2-fold increase in Vmaxc and Km values changed the model predicted amount of methanol in exhaled breath (AX) by a maximum of only 0.7%. The relative insensitivity of the model predicted exhaled breath levels of methanol is due to the fact that the blood/air partition coefficient value is large. Thus, once methanol is in systemic circulation, relatively little methanol is exhaled. Polar chemicals with large blood/air partition coefficient values, such as methanol, are amenable to a one-compartment analysis approach used by Perkins et al. (1995) to estimate RRU values for methanol in different species.
In this model, breathing rate (QP) was an important determinant for quantifying relative respiratory uptake of methanol. For example with QP values of 28.7, 38.7, and 48.7 l/h, the predicted amount of [14C]-methanol in exhaled breath was 41.6, 56, and 70.3 mg, respectively. Increasing the QP value from 28.7 to 48.7 l/h only changed the mixed venous blood concentrations from 1.9 to 2.3 mg/l.
Horton et al. (1992) developed a methanol model for young male rhesus monkeys (n = 3). The authors conducted whole-body, 6-h methanol-vapor exposures at either 50, 200, 1200, or 2000 ppm. Blood time-course data was collected for methanol after cessation of the methanol-vapor exposures. Dorman et al. (1994) conducted 2-h [14C]-methanol-vapor exposures in 4 adult female Cynomolgus monkeys under light anesthesia, using an endotracheal tube and a non-rebreathing valve. [14C]-Methanol-vapor exposure concentrations were 10, 45, 200, and 900 ppm. Inhaled and exhaled [14C]-methanol was measured in breath and blood during and after the 2-h [14C]-methanol vapor exposures. The Horton model was able to predict systemic clearance of methanol from blood after cessation of methanol-vapor exposure by adjusting metabolic rate-constant values until agreement was obtained between experimental data and simulation. Fractional uptake of inhaled methanol was not explicitly described in the Horton model structure. However, successful model predictions were achieved because metabolic clearance was assumed to be the primary biological determinant responsible for observed kinetics of methanol. This PBPK model suggests that 2 biological determinants are important for describing the systemic kinetics of inhaled methanol: fractional uptake of methanol vapors and metabolism of methanol.
The "wash-inwash-out" phenomenon for methanol vapors is an important consideration for risk assessment of methanol vapors. For example, the bioavailability of inhaled methanol vapor in lung was estimated to range from 40 to 81% of the actual vapor-exposure concentration in anesthetized female Cynomolgus monkeys. Consequently, the absorbed dose of methanol vapors would be overestimated, if the "wash-inwash-out" phenomenon were not to be considered. Also, the use of oral administration studies (e.g., drinking water or bolus gavage) in assessing the toxicology of inhaled methanol vapors may be inaccurate, because the bioavailability of methanol in the lung is less than the gut and first-pass metabolism further complicates the portal-of-entry comparison. This PBPK analysis of methanol vapors supports the use of simply adjusting the inhaled concentration to account for the "wash-inwash-out" of polar solvents, because the kinetic behavior of desorbed or "washed-out" methanol from the lung is linear.
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ACKNOWLEDGMENTS |
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NOTES |
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