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Blood ethanol concentration profiles: a comparison between rats and mice
Alcohol 29 (2003) 165–171
Blood ethanol concentration profiles: a comparison between rats and mice Daniel J. Livya, Scott E. Parnellb, James R. Westb,* a
Division of Anatomy, Faculty of Medicine and Dentistry, 5.01 Medical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2H7 Department of Human Anatomy and Medical Neurobiology, College of Medicine, The Texas A&M University System Health Science Center, College Station, TX 77843-1114, USA Received 22 February 2002; received in revised form 17 February 2003; accepted 17 February 2003
b
Abstract It is important to select an appropriate model system for studies examining the mechanisms of ethanol-induced injury. The most common model systems use either mice or rats with ethanol administered by means of intragastric gavage or intraperitoneal injection, yet few studies have compared directly the blood ethanol concentration (BEC) profiles that result from each of these model systems. In the current study, Sprague–Dawley rats and C57BL/6J mice were given ethanol by means of intragastric gavage or intraperitoneal injection at 40 days of age. Blood samples were collected at consistent time intervals to determine BECs. Blood ethanol concentrations in mice were sharper, with a more rapid rise to a sharp peak BEC, followed by a relatively rapid decline. In contrast, rat BEC profiles showed an initial rapid rise, followed by a more gradual rise to peak concentrations, and, then, a relatively gradual decline. This difference was particularly evident in rats receiving ethanol intragastrically. The differences found in BEC profiles between rats and mice and between ethanol administration paradigms may yield differences in the extent or mechanism of damage induced by ethanol, an important consideration when selecting an appropriate model for the investigation of ethanol-induced tissue damage. 쑖 2003 Elsevier Inc. All rights reserved. Keywords: Ethanol; Blood alcohol concentration; Rate of increase; Rate of elimination; Rat; Mouse
1. Introduction The choice of an appropriate animal model system for questions addressing the mechanisms of ethanol-induced injury should rely, at least in part, on the availability of accurate information about the differential responses of various species to ethanol exposure. Most studies that are designed to examine the mechanisms of actions of ethanol are performed in rats and mice. For many experiments, the choice between these two animal model systems often depends on the specific practical advantages that each offers, such as housing considerations, cost, or genetic line availability. In the past, the Sprague–Dawley rat model system was used by us or other investigators from our laboratory to examine the effects of ethanol administration on brain development. It was determined that peak blood ethanol concentration (BEC) is the best predictor of the severity of
* Corresponding author. Tel.: ⫹1-979-845-4991; fax: ⫹1-979-8450790. E-mail address: [email protected] (J.R. West). Editor: T.R. Jerrells 0741-8329/03/$ – see front matter 쑖 2003 Elsevier Inc. All rights reserved. doi: 10.1016/S0741-8329(03)00025-9
brain injury after exposure to ethanol during brain development (Bonthius & West, 1988; Pierce & West, 1986) and that ethanol, administered in a bingelike manner, produces a greater amount of damage to the developing brain when compared with findings for continuous exposure (Bonthius et al., 1988; Bonthius & West, 1990). We have recently incorporated mice into some of our experimental paradigms and were therefore interested in how BEC profiles in mice differed from those in the rat while variables, such as age of animal and dose and route of administration for ethanol, were controlled experimentally. Surprisingly, there are few reports contrasting the pharmacokinetics of ethanol in mice and rats, yet such differences could substantially affect the interpretation of cross-species comparisons. Abel (1982) reported that mice metabolize ethanol at a rate of 550 mg/kg/h; rats, at a rate of 300 mg/ kg/h; and human beings, at a rate of 100 mg/kg/h. Pastino et al. (1997) compared the pharmacokinetics of inhaled ethanol between rats and mice to find that BECs rose higher and faster in mice than in rats. However, it should be noted that inhalation is a relatively uncommon mode of ethanol administration and relies on selectively different mechanisms to offload the ethanol. Results from many other studies have
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included BEC profiles in either rats or mice, but not their contrast. Although comparisons among these different studies might prove revealing, the interpretation of such comparisons would be limited because of the different methods used in each. For example, many investigators report only the dose of ethanol administered and not the BEC achieved. However, the dose of ethanol administered does not accurately reflect how that ethanol was processed by each individual and is therefore an ineffective method of assessing the effects of ethanol. Those investigators that do report BECs often use different techniques to obtain their reported values (e.g., enzymatic degradation vs. chromatography). The methods used in other studies vary with respect to the location from which blood is sampled (e.g., tail vs. orbit), time of day at which ethanol is administered, and time at which food is removed from the animals. For these reasons, a direct comparison between species while all experimental parameters are held constant would seem a better way to contrast differential responses to ethanol. In the current study, BEC profiles were compared between adolescent Sprague–Dawley rats and C57BL/6J mice. Adolescent-aged animals were chosen because of the alarming increase in ethanol consumption by children, adolescents, and young adults (Alcohol use and abuse, 2001; DeWit et al., 2000; Sutherland & Shepherd, 2001), as well as evidence supporting the suggestion that ethanol exposure during this period may have serious consequences for the successful development of future offspring (Livy et al., 2001). Comparison of BEC profiles between these two species will allow a better understanding of the pharmacokinetic differences in their blood ethanol availability, which will better enable their relative choice as a model in studies of ethanol-induced developmental damage.
2. Methods 2.1. Animals Adolescent (40 days of age) Sprague–Dawley rats and C57BL/6J mice were used in this study. Animals were originally obtained from Harlan Laboratories (Indianapolis, IN; 35-day-old rats) and The Jackson Laboratory (Bar Harbor, ME; 35-day-old mice), or they were bred and reared at the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) International–accredited facility at Texas A&M University. All experimental protocols were approved by the Texas A&M University IACUC. All animals were reared on a normal 12-h light/12-h dark cycle and allowed free access to food and water except for a 2-h period of food deprivation before dosing. 2.2. Ethanol administration Both rats and mice received ethanol administered as a single dose of 3.8-g/kg of a 21.0% [weight/volume (wt./ vol.)] ethanol solution in sterile water either by means of
intragastric gavage (i.g.) or intraperitoneal injection (i.p.) at 2.5 h after the onset of the light cycle. Solution volumes administered to rats ranged from 2.5 to 3.0 ml (i.p.) and 2.5 to 2.9 ml (i.g.), whereas those administered to mice ranged from 0.28 to 0.41 ml (i.p.) and 0.28 to 0.42 ml (i.g.). A total of 20 rats were included, with 5 male and 5 female rats for each intraperitoneal and intragastric treatment condition. For mice, 10 male and 7 female mice received ethanol intraperitoneally, and 10 male and 8 female mice received ethanol intragastrically. Injected ethanol was delivered to both rats and mice by using a 26-gauge × 10-mm needle. Gavaged ethanol was delivered to mice by using a straight 22-gauge × 38-mm stainless steel feeding tube with a 1.25mm ball and to rats by using a curved 16-gauge × 76-mm stainless steel feeding tube with a 3.0-mm ball (Popper and Sons, Inc., New Hyde Park, NY) lubricated with a small drop of corn oil to facilitate passage down the esophagus. All animals were deprived of food for 2 h before administration of ethanol but retained free access to water. Tail blood samples were collected at 30, 60, 90, 120, 180, 270, 360, and 450 min after ethanol delivery. Samples were collected in 20-µl heparinized capillary tubes (Drummond Scientific Company, Broomall, PA) and transferred to 2-ml gas chromatographic vials containing 200 µl of an internal standard solution consisting of 0.6 N perchloric acid and 4 mM n-propanol in double distilled water. Vials were septum-sealed and stored at room temperature until analyzed by head space gas chromatography (Varian model 3900, Varian Medical Systems, Palo Alto, CA; Penton, 1987) to determine BEC. 2.3. Statistical analyses Blood ethanol concentration profiles were analyzed by using repeated-measures analysis of variance (ANOVA), contrasting species (rat vs. mouse) with administration method (intragastric gavage vs. intraperitoneal injection). In addition, two-way ANOVAs were used to analyze specific characteristics of the curves, including the straight-line slope to the 30-min time point, the straight-line slope to the measured peak BEC, and the straight-line slope from the measured peak to the 360-min time point. It should be noted that peak BEC refers to the highest measured value. All statistics were performed by using Statview 5.0.
3. Results No effect of sex of animal was found for any of the measures, and therefore males and females were combined within each treatment group. Fig. 1 shows the mean BEC results at each of the time points for mice and rats given ethanol by both intraperitoneal injection and intragastric gavage paradigms. There was a significant interaction between species and administration route over the time course of blood sampling (P ⫽ .012), indicating that mice and rats
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Fig. 1. Blood ethanol concentration profiles for rats and mice that received ethanol by means of intragastric gavage or intraperitoneal injection. The mean and standard error are shown at each of the blood collection times. There was a significant (P ⫽ .012) interaction between species and administration route over the time of blood sampling. Blood ethanol concentration profiles in mice were sharper, with a rapid rise to an early peak concentration (about 60 min after administration), followed by a relatively rapid decline. In rats, the profile was more gradual, with an initial rapid rise, followed by a more gradual rise to a lower and later peak, and, then, a gradual decline. Note the relatively flat response to ethanol in rats receiving ethanol by intragastric gavage.
differ in their response to ethanol administered by means of intragastric gavage and intraperitoneal injection. As indicated in Fig. 1, mice showed a rapid increase in BEC to an average measured peak BEC at 60 min after ethanol administration by both intragastric gavage and intraperitoneal injection. This was followed by a relatively steep decline in BEC until a concentration close to zero was obtained at the 360-min sampling period. In contrast, injected rats showed a rapid rise in BEC during the first 30 min. The rate of BEC increase continued to rise until the average measured peak was reached at 120 min after administration, but at a greatly reduced rate. This was followed by a gradual, steady decline, but the BEC had not returned to zero by the 450min sampling point despite the lower measured peak concentrations relative to those attained by the mice. The gavaged rats showed a rapid rise in BEC during the first 30 min, but to a relatively low concentration (74 mg/dl). This was followed by a gradual rise to a measured peak at 60 min and then a gradual decline to a concentration close to zero at the 450-min sampling point. In both species, ethanol administered by means of intraperitoneal injection resulted in higher BECs than those observed for intragastric gavage. The slope of the line describing the initial rise in BEC provides a measure of the rate of ethanol absorption into the bloodstream. Fig. 2A shows the slope of BEC increase measured during the first
30 min (the time of largest increase). No interaction was found, but mice showed a more rapid increase in BEC than observed for rats (P ⬍ .0001), and the rate of BEC increase was greater for ethanol administered by means of intraperitoneal injection compared with findings for intragastric gavage administration (P ⬍ .0001). In addition, the slope of the BEC increase up to the time of the measured peak BEC was assessed. The results were very similar to those found for the first 30 min. As shown in Fig. 2B, no interaction was present, but mice did show a much larger increase in BEC during a shorter period than that shown in rats (P ⬍ .0001), and BECs showed a more rapid increase due to intraperitoneal versus intragastric ethanol administration (P ⫽ .0013). The rate of increase of BEC within an animal describes the rate of absorption of ethanol into the bloodstream. The slope of the decreasing BECs occurring after the peak BEC provides an estimate of the rate of ethanol metabolism within the bloodstream. This decreasing slope was calculated between the measured peak BEC and the 360-min BEC measurement. As shown in Fig. 2C, BECs dropped at a greater rate in mice as compared with findings for rats (P ⬍ .0001), and the rate of decline was again higher in those animals receiving ethanol by means of intraperitoneal injection (P ⫽ .0042). From the above analyses it can be seen that, in general, mice have an initial rapid increase in BEC to a relatively
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Fig. 2. Mean rate (and standard error) of ethanol concentration change. A) Mean rate of increase to the 30-min blood ethanol concentration (BEC). Blood ethanol concentrations increased more rapidly in mice than in rats (P ⬍ .0001) and in animals receiving ethanol by means of intraperitoneal injection versus intragastric gavage (P ⬍ .0001). B) Mean rate of increase to the measured peak BEC. Blood ethanol concentrations increased more rapidly in mice than in rats (P ⬍ .0001) and in animals receiving ethanol by means of intraperitoneal injection versus intragastric gavage (P ⫽ .0013). C) Mean rate of ethanol elimination from the peak BEC to the 360-min sampling period. Blood ethanol concentrations decreased more rapidly in mice than in rats (P ⬍ .0001) and in animals that received ethanol by means of intraperitoneal injection versus intragastric gavage (P ⫽ .0042).
high peak, followed by a rapid decline in BEC. In contrast, rats have a slower increase in BEC to a relatively low peak, and then a more gradual BEC decline. However, the rate of increase and decrease in the BEC does not reflect potential differences between mice and rats with respect to their total exposure to ethanol throughout the profile period. In other words, do the sharper slopes but higher BEC peaks experienced in the mice equate to the more gradual slopes but lower BEC peaks experienced in the rats?
4. Discussion The results of the current study indicate that adolescent rats and mice respond differently to ethanol administration and that the differences between them are modified by the mode of ethanol delivery. In general, BEC profiles in mice are sharper, with a more rapid rise to a sharp peak in BEC, followed by a relatively rapid decline. Blood ethanol concentration profiles in rats are less severe. They show an
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initial rapid rise, followed by a relatively prolonged and gradual increase to peak concentrations, and, then, a relatively gradual decline. The BECs reported in this article are similar to those reported in other published studies, relative to the inherent variability among treatment paradigms (Bonthius et al., 2002; Ogilvie et al., 1997; Ott et al., 1985; Pan & Hedaya, 1999; Webster et al., 1983). An exception may be the BECs obtained for gavaged rats, which were surprisingly low. The reason why these BECs are low is unclear, although it should be noted that the low standard errors support the suggestion that all animals responded in a similar manner. Intragastric ethanol administration produced significantly lower BECs than observed with intraperitoneal ethanol administration, a difference particularly evident in the rats. In injected animals, ethanol is absorbed directly from the peritoneal cavity into the portal bloodstream, where it travels to the liver and is metabolized by hepatic alcohol dehydrogenase (ADH). Gavaged animals display a similar mode of ethanol metabolism after the absorption of ethanol from the stomach and duodenum into the portal bloodstream. However, administration of ethanol into the stomach also permits a second source of metabolism to occur, by means of gastric ADH (Lim et al., 1993; Roine et al., 1990). This “extra” metabolism means that less ethanol is available for entry into the systemic bloodstream, resulting in lower BECs in the gavaged animals. Additional metabolic factors include catalase (Keilin & Hartree, 1945; Lieber, 1992; Smith, 1961) and the microsomal ethanol-oxidizing system (MEOS; Lieber & DeCarli, 1968, 1970). However, catalase is not thought to contribute significantly to ethanol metabolism under normal pharmacological conditions, and the MEOS is thought to be more involved with metabolism in cases of chronic ethanol exposure (Lieber, 1999). The disparate BEC profiles of rats and mice support the suggestion of differences in the rates of ethanol absorption into, and elimination from, the bloodstream. Because both rats and mice were given identical doses of ethanol by body weight, both should have had the same relative quantity of ethanol available for uptake into the systemic bloodstream. The rapid rise in mouse BECs displayed for both modes of ethanol delivery supports the suggestion of a faster rate of ethanol absorption into the bloodstream. In gavaged animals, the rate of this absorption depends, in part, on the rate of gastric emptying into the duodenum. Gastric emptying is slower in rats than in mice (Bossoni et al., 1979), supporting the suggestion that rats not only have a slower rate of ethanol delivery to the duodenum for absorption into the bloodstream, but also hold the ethanol in their stomach for a longer period. This allows the gastric ADH to metabolize more of the ethanol while in the stomach, making less ethanol available for entry into the duodenum. Therefore, less ethanol is available for absorption into the portal bloodstream, resulting in relatively lower BECs. Differences in the rate, or onset, of ethanol elimination may also affect the shape of BEC profiles. Ethanol metabolism will begin as soon as ethanol encounters ADH. Mice
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show a more rapid rate of ethanol absorption into the bloodstream and should therefore show a more rapid onset of metabolism. However, it is unlikely that this difference would account for the dramatic difference in BEC profiles between rats and mice. In comparison with rats, mice show a faster rate of ethanol elimination from the systemic circulation, which may reflect a faster rate of ethanol clearance from the portal circulation to the liver, thereby enabling a faster rate of diffusion into the portal circulation from the peritoneum, duodenum, or stomach. Alternatively, ADH efficiency may be greater in mice. Structurally, the rat class I ADH gene and the mouse ADH-1 gene are very similar (Crabb et al., 1989). However, this does not mean that their functional activities are the same. In addition, Balak et al. (1982) found that C57BL/6 mice synthesize hepatic ADH more rapidly than BALB/c mice do, supporting the suggestion of a greater availability of hepatic ADH in the C57BL/6 mice during the metabolic process. Such differences may be more pronounced in the rat, ultimately leading to their relatively lower rates of elimination. Blood ethanol concentrations did not differ significantly between the sexes. These results support those reported previously in younger animals (Kelly et al., 1987; Middaugh et al., 1992). Sex differences have been reported in adult animals (Desroches et al., 1995; Middaugh et al., 1992) and human beings (Baraona et al., 2001; Frezza et al., 1990; Lieber, 2000), although Seitz et al. (1993) suggested that such differences may be negated with advancing age. Blood ethanol concentration is an important factor when determining the potential for ethanol-induced tissue damage. Blood ethanol concentration is a more accurate indicator of the potential for ethanol-induced tissue damage than ethanol dose administered because of the metabolic differences among individuals (Bonthius et al., 1988). Indeed, Bonthius and West (1988) found that peak BEC is a critical factor when predicting the extent of damage caused by ethanol administration during the time of brain development. Another important factor may be the rate of BEC increase to the peak concentration. Desiderio (1987, 1988) found that the rate of BEC increase reduced significantly the survival of dogs after an induced cardiac injury. In the current study, in comparison with rats, mice achieved a higher peak BEC, despite equal dose administration, and achieved this concentration more rapidly. These are important factors when considering the choice between rats and mice as a model for the study of ethanolrelated brain damage during development. It should be noted that the results described in this article are specific to the conditions listed. Advancing maturity, previous exposure to ethanol, and pregnancy may affect the BEC response of an animal. For example, Kelly et al. (1987) reported an increase in ethanol elimination rates during the first 60 days of rat development. The results are also specific to the animal models described. Pastino et al. (1996) found that gastric ADH activity in male Swiss–Webster mice contributed negligibly to the reduction in BECs after oral ethanol consumption. In contrast, gastric ADH seems to play a more
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significant role in the metabolism of ethanol in C57BL/6J mice, although certainly not to the extent as that shown in the Sprague–Dawley rats. It is important to acknowledge the limitations of these animal models as representatives of similar conditions in human beings. Ethanol consumption in human beings usually occurs by self-administered drinking involving multiple dosing over a relatively extended period. Allowing animals free access to self-delivered ethanol does not provide adequate control of amount consumed or timing of consumption. The gavage model permits this extra degree of experimental control while retaining the physiological relevance of oral delivery, but it still involves a degree of compromise from the paradigm of human consumption. In human beings, ethanol is first introduced into the oral cavity and then swallowed. While in the mouth, some of the ethanol is absorbed rapidly through the rich blood supply of the buccal mucosa (Bourne et al., 1986). This ethanol bypasses the portal circulation, thus delaying its metabolism. This results in higher relative BECs than those observed with the same amount of ethanol delivered by means of gavage. In addition, the controlled delivery of ethanol to animals usually involves some form of restraint to permit the delivery in an efficient manner. Some amount of stress is inherent with any method of restraint and may also affect the response of the animal to the ethanol delivered. Despite these differences, ethanol administration by means of gavage is arguably a better method for ethanol delivery while retaining experimental control and physiological relevance. We have demonstrated significant differences in ethanol response between mice and rats. Mice show rapid rise and elimination rates and higher peak BECs. Rats show more gradual profiles but retain the ethanol in their bloodstream for longer periods. Gavage administration highlighted these differences. The relatively flat BEC profile experienced by the rat may be an important consideration when evaluating the overall exposure of the brain to ethanol. Because various species can metabolize a given dose of ethanol at significantly different rates, the peak BEC may be one important way to compare comparable exposure levels. Therefore, the reported differences between rats and mice emphasize the importance of making an informed choice when selecting the appropriate model for use in the study of ethanol effects on brain development. Acknowledgments We wish to acknowledge the assistance of Ms. Kathryn T. Gutierrez and Ms. Cassie Behrendt with animal handling. This work was supported in part by grants AA10090 and AA05523 from the National Institute on Alcohol Abuse and Alcoholism (to JRW). References Abel, E. L. (1982). Behavioral teratology of alcohol (animal model studies of the fetal alcohol syndrome). In E. L. Abel (Ed.), Fetal Alcohol
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Blood ethanol concentration profiles: a comparison between rats and mice Daniel J. Livya, Scott E. Parnellb, James R. Westb,* a
Division of Anatomy, Faculty of Medicine and Dentistry, 5.01 Medical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2H7 Department of Human Anatomy and Medical Neurobiology, College of Medicine, The Texas A&M University System Health Science Center, College Station, TX 77843-1114, USA Received 22 February 2002; received in revised form 17 February 2003; accepted 17 February 2003
b
Abstract It is important to select an appropriate model system for studies examining the mechanisms of ethanol-induced injury. The most common model systems use either mice or rats with ethanol administered by means of intragastric gavage or intraperitoneal injection, yet few studies have compared directly the blood ethanol concentration (BEC) profiles that result from each of these model systems. In the current study, Sprague–Dawley rats and C57BL/6J mice were given ethanol by means of intragastric gavage or intraperitoneal injection at 40 days of age. Blood samples were collected at consistent time intervals to determine BECs. Blood ethanol concentrations in mice were sharper, with a more rapid rise to a sharp peak BEC, followed by a relatively rapid decline. In contrast, rat BEC profiles showed an initial rapid rise, followed by a more gradual rise to peak concentrations, and, then, a relatively gradual decline. This difference was particularly evident in rats receiving ethanol intragastrically. The differences found in BEC profiles between rats and mice and between ethanol administration paradigms may yield differences in the extent or mechanism of damage induced by ethanol, an important consideration when selecting an appropriate model for the investigation of ethanol-induced tissue damage. 쑖 2003 Elsevier Inc. All rights reserved. Keywords: Ethanol; Blood alcohol concentration; Rate of increase; Rate of elimination; Rat; Mouse
1. Introduction The choice of an appropriate animal model system for questions addressing the mechanisms of ethanol-induced injury should rely, at least in part, on the availability of accurate information about the differential responses of various species to ethanol exposure. Most studies that are designed to examine the mechanisms of actions of ethanol are performed in rats and mice. For many experiments, the choice between these two animal model systems often depends on the specific practical advantages that each offers, such as housing considerations, cost, or genetic line availability. In the past, the Sprague–Dawley rat model system was used by us or other investigators from our laboratory to examine the effects of ethanol administration on brain development. It was determined that peak blood ethanol concentration (BEC) is the best predictor of the severity of
* Corresponding author. Tel.: ⫹1-979-845-4991; fax: ⫹1-979-8450790. E-mail address: [email protected] (J.R. West). Editor: T.R. Jerrells 0741-8329/03/$ – see front matter 쑖 2003 Elsevier Inc. All rights reserved. doi: 10.1016/S0741-8329(03)00025-9
brain injury after exposure to ethanol during brain development (Bonthius & West, 1988; Pierce & West, 1986) and that ethanol, administered in a bingelike manner, produces a greater amount of damage to the developing brain when compared with findings for continuous exposure (Bonthius et al., 1988; Bonthius & West, 1990). We have recently incorporated mice into some of our experimental paradigms and were therefore interested in how BEC profiles in mice differed from those in the rat while variables, such as age of animal and dose and route of administration for ethanol, were controlled experimentally. Surprisingly, there are few reports contrasting the pharmacokinetics of ethanol in mice and rats, yet such differences could substantially affect the interpretation of cross-species comparisons. Abel (1982) reported that mice metabolize ethanol at a rate of 550 mg/kg/h; rats, at a rate of 300 mg/ kg/h; and human beings, at a rate of 100 mg/kg/h. Pastino et al. (1997) compared the pharmacokinetics of inhaled ethanol between rats and mice to find that BECs rose higher and faster in mice than in rats. However, it should be noted that inhalation is a relatively uncommon mode of ethanol administration and relies on selectively different mechanisms to offload the ethanol. Results from many other studies have
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included BEC profiles in either rats or mice, but not their contrast. Although comparisons among these different studies might prove revealing, the interpretation of such comparisons would be limited because of the different methods used in each. For example, many investigators report only the dose of ethanol administered and not the BEC achieved. However, the dose of ethanol administered does not accurately reflect how that ethanol was processed by each individual and is therefore an ineffective method of assessing the effects of ethanol. Those investigators that do report BECs often use different techniques to obtain their reported values (e.g., enzymatic degradation vs. chromatography). The methods used in other studies vary with respect to the location from which blood is sampled (e.g., tail vs. orbit), time of day at which ethanol is administered, and time at which food is removed from the animals. For these reasons, a direct comparison between species while all experimental parameters are held constant would seem a better way to contrast differential responses to ethanol. In the current study, BEC profiles were compared between adolescent Sprague–Dawley rats and C57BL/6J mice. Adolescent-aged animals were chosen because of the alarming increase in ethanol consumption by children, adolescents, and young adults (Alcohol use and abuse, 2001; DeWit et al., 2000; Sutherland & Shepherd, 2001), as well as evidence supporting the suggestion that ethanol exposure during this period may have serious consequences for the successful development of future offspring (Livy et al., 2001). Comparison of BEC profiles between these two species will allow a better understanding of the pharmacokinetic differences in their blood ethanol availability, which will better enable their relative choice as a model in studies of ethanol-induced developmental damage.
2. Methods 2.1. Animals Adolescent (40 days of age) Sprague–Dawley rats and C57BL/6J mice were used in this study. Animals were originally obtained from Harlan Laboratories (Indianapolis, IN; 35-day-old rats) and The Jackson Laboratory (Bar Harbor, ME; 35-day-old mice), or they were bred and reared at the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) International–accredited facility at Texas A&M University. All experimental protocols were approved by the Texas A&M University IACUC. All animals were reared on a normal 12-h light/12-h dark cycle and allowed free access to food and water except for a 2-h period of food deprivation before dosing. 2.2. Ethanol administration Both rats and mice received ethanol administered as a single dose of 3.8-g/kg of a 21.0% [weight/volume (wt./ vol.)] ethanol solution in sterile water either by means of
intragastric gavage (i.g.) or intraperitoneal injection (i.p.) at 2.5 h after the onset of the light cycle. Solution volumes administered to rats ranged from 2.5 to 3.0 ml (i.p.) and 2.5 to 2.9 ml (i.g.), whereas those administered to mice ranged from 0.28 to 0.41 ml (i.p.) and 0.28 to 0.42 ml (i.g.). A total of 20 rats were included, with 5 male and 5 female rats for each intraperitoneal and intragastric treatment condition. For mice, 10 male and 7 female mice received ethanol intraperitoneally, and 10 male and 8 female mice received ethanol intragastrically. Injected ethanol was delivered to both rats and mice by using a 26-gauge × 10-mm needle. Gavaged ethanol was delivered to mice by using a straight 22-gauge × 38-mm stainless steel feeding tube with a 1.25mm ball and to rats by using a curved 16-gauge × 76-mm stainless steel feeding tube with a 3.0-mm ball (Popper and Sons, Inc., New Hyde Park, NY) lubricated with a small drop of corn oil to facilitate passage down the esophagus. All animals were deprived of food for 2 h before administration of ethanol but retained free access to water. Tail blood samples were collected at 30, 60, 90, 120, 180, 270, 360, and 450 min after ethanol delivery. Samples were collected in 20-µl heparinized capillary tubes (Drummond Scientific Company, Broomall, PA) and transferred to 2-ml gas chromatographic vials containing 200 µl of an internal standard solution consisting of 0.6 N perchloric acid and 4 mM n-propanol in double distilled water. Vials were septum-sealed and stored at room temperature until analyzed by head space gas chromatography (Varian model 3900, Varian Medical Systems, Palo Alto, CA; Penton, 1987) to determine BEC. 2.3. Statistical analyses Blood ethanol concentration profiles were analyzed by using repeated-measures analysis of variance (ANOVA), contrasting species (rat vs. mouse) with administration method (intragastric gavage vs. intraperitoneal injection). In addition, two-way ANOVAs were used to analyze specific characteristics of the curves, including the straight-line slope to the 30-min time point, the straight-line slope to the measured peak BEC, and the straight-line slope from the measured peak to the 360-min time point. It should be noted that peak BEC refers to the highest measured value. All statistics were performed by using Statview 5.0.
3. Results No effect of sex of animal was found for any of the measures, and therefore males and females were combined within each treatment group. Fig. 1 shows the mean BEC results at each of the time points for mice and rats given ethanol by both intraperitoneal injection and intragastric gavage paradigms. There was a significant interaction between species and administration route over the time course of blood sampling (P ⫽ .012), indicating that mice and rats
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Fig. 1. Blood ethanol concentration profiles for rats and mice that received ethanol by means of intragastric gavage or intraperitoneal injection. The mean and standard error are shown at each of the blood collection times. There was a significant (P ⫽ .012) interaction between species and administration route over the time of blood sampling. Blood ethanol concentration profiles in mice were sharper, with a rapid rise to an early peak concentration (about 60 min after administration), followed by a relatively rapid decline. In rats, the profile was more gradual, with an initial rapid rise, followed by a more gradual rise to a lower and later peak, and, then, a gradual decline. Note the relatively flat response to ethanol in rats receiving ethanol by intragastric gavage.
differ in their response to ethanol administered by means of intragastric gavage and intraperitoneal injection. As indicated in Fig. 1, mice showed a rapid increase in BEC to an average measured peak BEC at 60 min after ethanol administration by both intragastric gavage and intraperitoneal injection. This was followed by a relatively steep decline in BEC until a concentration close to zero was obtained at the 360-min sampling period. In contrast, injected rats showed a rapid rise in BEC during the first 30 min. The rate of BEC increase continued to rise until the average measured peak was reached at 120 min after administration, but at a greatly reduced rate. This was followed by a gradual, steady decline, but the BEC had not returned to zero by the 450min sampling point despite the lower measured peak concentrations relative to those attained by the mice. The gavaged rats showed a rapid rise in BEC during the first 30 min, but to a relatively low concentration (74 mg/dl). This was followed by a gradual rise to a measured peak at 60 min and then a gradual decline to a concentration close to zero at the 450-min sampling point. In both species, ethanol administered by means of intraperitoneal injection resulted in higher BECs than those observed for intragastric gavage. The slope of the line describing the initial rise in BEC provides a measure of the rate of ethanol absorption into the bloodstream. Fig. 2A shows the slope of BEC increase measured during the first
30 min (the time of largest increase). No interaction was found, but mice showed a more rapid increase in BEC than observed for rats (P ⬍ .0001), and the rate of BEC increase was greater for ethanol administered by means of intraperitoneal injection compared with findings for intragastric gavage administration (P ⬍ .0001). In addition, the slope of the BEC increase up to the time of the measured peak BEC was assessed. The results were very similar to those found for the first 30 min. As shown in Fig. 2B, no interaction was present, but mice did show a much larger increase in BEC during a shorter period than that shown in rats (P ⬍ .0001), and BECs showed a more rapid increase due to intraperitoneal versus intragastric ethanol administration (P ⫽ .0013). The rate of increase of BEC within an animal describes the rate of absorption of ethanol into the bloodstream. The slope of the decreasing BECs occurring after the peak BEC provides an estimate of the rate of ethanol metabolism within the bloodstream. This decreasing slope was calculated between the measured peak BEC and the 360-min BEC measurement. As shown in Fig. 2C, BECs dropped at a greater rate in mice as compared with findings for rats (P ⬍ .0001), and the rate of decline was again higher in those animals receiving ethanol by means of intraperitoneal injection (P ⫽ .0042). From the above analyses it can be seen that, in general, mice have an initial rapid increase in BEC to a relatively
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Fig. 2. Mean rate (and standard error) of ethanol concentration change. A) Mean rate of increase to the 30-min blood ethanol concentration (BEC). Blood ethanol concentrations increased more rapidly in mice than in rats (P ⬍ .0001) and in animals receiving ethanol by means of intraperitoneal injection versus intragastric gavage (P ⬍ .0001). B) Mean rate of increase to the measured peak BEC. Blood ethanol concentrations increased more rapidly in mice than in rats (P ⬍ .0001) and in animals receiving ethanol by means of intraperitoneal injection versus intragastric gavage (P ⫽ .0013). C) Mean rate of ethanol elimination from the peak BEC to the 360-min sampling period. Blood ethanol concentrations decreased more rapidly in mice than in rats (P ⬍ .0001) and in animals that received ethanol by means of intraperitoneal injection versus intragastric gavage (P ⫽ .0042).
high peak, followed by a rapid decline in BEC. In contrast, rats have a slower increase in BEC to a relatively low peak, and then a more gradual BEC decline. However, the rate of increase and decrease in the BEC does not reflect potential differences between mice and rats with respect to their total exposure to ethanol throughout the profile period. In other words, do the sharper slopes but higher BEC peaks experienced in the mice equate to the more gradual slopes but lower BEC peaks experienced in the rats?
4. Discussion The results of the current study indicate that adolescent rats and mice respond differently to ethanol administration and that the differences between them are modified by the mode of ethanol delivery. In general, BEC profiles in mice are sharper, with a more rapid rise to a sharp peak in BEC, followed by a relatively rapid decline. Blood ethanol concentration profiles in rats are less severe. They show an
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initial rapid rise, followed by a relatively prolonged and gradual increase to peak concentrations, and, then, a relatively gradual decline. The BECs reported in this article are similar to those reported in other published studies, relative to the inherent variability among treatment paradigms (Bonthius et al., 2002; Ogilvie et al., 1997; Ott et al., 1985; Pan & Hedaya, 1999; Webster et al., 1983). An exception may be the BECs obtained for gavaged rats, which were surprisingly low. The reason why these BECs are low is unclear, although it should be noted that the low standard errors support the suggestion that all animals responded in a similar manner. Intragastric ethanol administration produced significantly lower BECs than observed with intraperitoneal ethanol administration, a difference particularly evident in the rats. In injected animals, ethanol is absorbed directly from the peritoneal cavity into the portal bloodstream, where it travels to the liver and is metabolized by hepatic alcohol dehydrogenase (ADH). Gavaged animals display a similar mode of ethanol metabolism after the absorption of ethanol from the stomach and duodenum into the portal bloodstream. However, administration of ethanol into the stomach also permits a second source of metabolism to occur, by means of gastric ADH (Lim et al., 1993; Roine et al., 1990). This “extra” metabolism means that less ethanol is available for entry into the systemic bloodstream, resulting in lower BECs in the gavaged animals. Additional metabolic factors include catalase (Keilin & Hartree, 1945; Lieber, 1992; Smith, 1961) and the microsomal ethanol-oxidizing system (MEOS; Lieber & DeCarli, 1968, 1970). However, catalase is not thought to contribute significantly to ethanol metabolism under normal pharmacological conditions, and the MEOS is thought to be more involved with metabolism in cases of chronic ethanol exposure (Lieber, 1999). The disparate BEC profiles of rats and mice support the suggestion of differences in the rates of ethanol absorption into, and elimination from, the bloodstream. Because both rats and mice were given identical doses of ethanol by body weight, both should have had the same relative quantity of ethanol available for uptake into the systemic bloodstream. The rapid rise in mouse BECs displayed for both modes of ethanol delivery supports the suggestion of a faster rate of ethanol absorption into the bloodstream. In gavaged animals, the rate of this absorption depends, in part, on the rate of gastric emptying into the duodenum. Gastric emptying is slower in rats than in mice (Bossoni et al., 1979), supporting the suggestion that rats not only have a slower rate of ethanol delivery to the duodenum for absorption into the bloodstream, but also hold the ethanol in their stomach for a longer period. This allows the gastric ADH to metabolize more of the ethanol while in the stomach, making less ethanol available for entry into the duodenum. Therefore, less ethanol is available for absorption into the portal bloodstream, resulting in relatively lower BECs. Differences in the rate, or onset, of ethanol elimination may also affect the shape of BEC profiles. Ethanol metabolism will begin as soon as ethanol encounters ADH. Mice
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show a more rapid rate of ethanol absorption into the bloodstream and should therefore show a more rapid onset of metabolism. However, it is unlikely that this difference would account for the dramatic difference in BEC profiles between rats and mice. In comparison with rats, mice show a faster rate of ethanol elimination from the systemic circulation, which may reflect a faster rate of ethanol clearance from the portal circulation to the liver, thereby enabling a faster rate of diffusion into the portal circulation from the peritoneum, duodenum, or stomach. Alternatively, ADH efficiency may be greater in mice. Structurally, the rat class I ADH gene and the mouse ADH-1 gene are very similar (Crabb et al., 1989). However, this does not mean that their functional activities are the same. In addition, Balak et al. (1982) found that C57BL/6 mice synthesize hepatic ADH more rapidly than BALB/c mice do, supporting the suggestion of a greater availability of hepatic ADH in the C57BL/6 mice during the metabolic process. Such differences may be more pronounced in the rat, ultimately leading to their relatively lower rates of elimination. Blood ethanol concentrations did not differ significantly between the sexes. These results support those reported previously in younger animals (Kelly et al., 1987; Middaugh et al., 1992). Sex differences have been reported in adult animals (Desroches et al., 1995; Middaugh et al., 1992) and human beings (Baraona et al., 2001; Frezza et al., 1990; Lieber, 2000), although Seitz et al. (1993) suggested that such differences may be negated with advancing age. Blood ethanol concentration is an important factor when determining the potential for ethanol-induced tissue damage. Blood ethanol concentration is a more accurate indicator of the potential for ethanol-induced tissue damage than ethanol dose administered because of the metabolic differences among individuals (Bonthius et al., 1988). Indeed, Bonthius and West (1988) found that peak BEC is a critical factor when predicting the extent of damage caused by ethanol administration during the time of brain development. Another important factor may be the rate of BEC increase to the peak concentration. Desiderio (1987, 1988) found that the rate of BEC increase reduced significantly the survival of dogs after an induced cardiac injury. In the current study, in comparison with rats, mice achieved a higher peak BEC, despite equal dose administration, and achieved this concentration more rapidly. These are important factors when considering the choice between rats and mice as a model for the study of ethanolrelated brain damage during development. It should be noted that the results described in this article are specific to the conditions listed. Advancing maturity, previous exposure to ethanol, and pregnancy may affect the BEC response of an animal. For example, Kelly et al. (1987) reported an increase in ethanol elimination rates during the first 60 days of rat development. The results are also specific to the animal models described. Pastino et al. (1996) found that gastric ADH activity in male Swiss–Webster mice contributed negligibly to the reduction in BECs after oral ethanol consumption. In contrast, gastric ADH seems to play a more
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significant role in the metabolism of ethanol in C57BL/6J mice, although certainly not to the extent as that shown in the Sprague–Dawley rats. It is important to acknowledge the limitations of these animal models as representatives of similar conditions in human beings. Ethanol consumption in human beings usually occurs by self-administered drinking involving multiple dosing over a relatively extended period. Allowing animals free access to self-delivered ethanol does not provide adequate control of amount consumed or timing of consumption. The gavage model permits this extra degree of experimental control while retaining the physiological relevance of oral delivery, but it still involves a degree of compromise from the paradigm of human consumption. In human beings, ethanol is first introduced into the oral cavity and then swallowed. While in the mouth, some of the ethanol is absorbed rapidly through the rich blood supply of the buccal mucosa (Bourne et al., 1986). This ethanol bypasses the portal circulation, thus delaying its metabolism. This results in higher relative BECs than those observed with the same amount of ethanol delivered by means of gavage. In addition, the controlled delivery of ethanol to animals usually involves some form of restraint to permit the delivery in an efficient manner. Some amount of stress is inherent with any method of restraint and may also affect the response of the animal to the ethanol delivered. Despite these differences, ethanol administration by means of gavage is arguably a better method for ethanol delivery while retaining experimental control and physiological relevance. We have demonstrated significant differences in ethanol response between mice and rats. Mice show rapid rise and elimination rates and higher peak BECs. Rats show more gradual profiles but retain the ethanol in their bloodstream for longer periods. Gavage administration highlighted these differences. The relatively flat BEC profile experienced by the rat may be an important consideration when evaluating the overall exposure of the brain to ethanol. Because various species can metabolize a given dose of ethanol at significantly different rates, the peak BEC may be one important way to compare comparable exposure levels. Therefore, the reported differences between rats and mice emphasize the importance of making an informed choice when selecting the appropriate model for use in the study of ethanol effects on brain development. Acknowledgments We wish to acknowledge the assistance of Ms. Kathryn T. Gutierrez and Ms. Cassie Behrendt with animal handling. This work was supported in part by grants AA10090 and AA05523 from the National Institute on Alcohol Abuse and Alcoholism (to JRW). References Abel, E. L. (1982). Behavioral teratology of alcohol (animal model studies of the fetal alcohol syndrome). In E. L. Abel (Ed.), Fetal Alcohol
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