The Role of Monocarboxylate Transporters and Their Chaperone CD147 in Lactate Efflux Inhibition and the Anticancer Effects of Terminalia chebula in Neuroblastoma Cell Line N2-A.

AIMS: In the presence of oxygen, most of the synthesized pyruvate during glycolysis in the cancer cell of solid tumors is released away from the mitochondria to form lactate (Warburg Effect). To maintain cell homeostasis, lactate is transported across the cell membrane by monocarboxylate transporters (MCTs). The major aim of the current investigation is to identify novel compounds that inhibit lactate efflux that may lead to identifying effective targets for cancer treatment. STUDY DESIGN: In this study, 900 ethanol plant extracts were screened for their lactate efflux inhibition using neuroblastoma (N2-A) cell line. Additionally, we investigated the mechanism of inhibition for the most potent plant extract regarding monocarboxylate transporters expression, and consequences effects on viability, growth, and apoptosis. METHODOLOGY: The potency of lactate efflux inhibition of ethanol plant extracts was evaluated in N2-A cells by measuring extracellular lactate levels. Caspase 3- activity and acridine orange/ethidium bromide staining were performed to assess the apoptotic effect. The antiproliferative effect was measured using WST assay. Western blotting was performed to quantify protein expression of MCTs and their chaperone CD147 in treated cells lysates. RESULTS: Terminalia chebula plant extract was the most potent lactate efflux inhibitor in N2-A cells among the 900 - tested plant extracts. The results obtained show that extract of Terminalia chebula fruits (TCE) significantly (P = 0.05) reduced the expression of the MCT1, MCT3, MCT4 and the chaperone CD147. The plant extract was more potent (IC50 of 3.59 ± 0.26 µg/ml) than the MCT standard inhibitor phloretin (IC50 76.54 ± 3.19 µg/ml). The extract also showed more potency and selective cytotoxicity in cancer cells than DI-TNC1 primary cell line (IC50 7.37 ± 0.28 vs. 17.35 ± 0.19 µg/ml). Moreover, TCE Inhibited N2-A cell growth (IG50 = 5.20 ± 0.30 µg/ml) and induced apoptosis at the 7.5 µg/ml concentration. CONCLUSION: Out of the 900 plant extracts screened, Terminalia chebula ethanol extract was found to be the most potent lactate efflux inhibitor with the ability to inhibit chaperone CD147 expression and impact the function of monocarboxylate transporters. Furthermore, TCE was found to have growth inhibition and apoptotic effects. The results obtained indicate that Terminalia chebula constituent(s) may contain promising compounds that can be useful in the management of neuroblastoma cancer.

Streptozocin-induced diabetes affects rat urinary bladder response to autonomic agents.

The response of the urinary bladder body and base to autonomic agents was studied in streptozocin (STZ)-diabetic rats. The bladder body region from 6-wk diabetic rats showed no changes in response to acetylcholine, phenylephrine, or isoproterenol. In contrast, the bladder base region showed a 39% increase in contractile response to acetylcholine and a 37% increased response to phenylephrine. In tissues from 47-wk diabetic animals, the bladder body showed a 51% increased contractile response to acetylcholine and a 37% increased relaxation response to isoproterenol. The bladder base showed a 66% increased contraction to acetylcholine. Thus, in the bladder base, enhanced responses to acetylcholine are detected soon after induction of diabetes and continue to increase as the diabetic state progresses. Moreover, in the same bladder region, an increase in responsiveness to alpha-adrenergic stimuli occurs. In the bladder body, enhanced responses to cholinergic and to beta-adrenergic stimuli occur, but are only observed in a more chronic diabetic state. The data suggest that an effect associated with autonomic diabetic neuropathy of the urinary bladder is an increased postsynaptic responsiveness to cholinergic stimuli in both regions.

Age-related changes in sensitivity of rat urinary bladder to autonomic agents.

Experiments were done to determine if age-related changes occur in autonomic regulation of rat urinary bladder. The maximum contractile responses to acetylcholine were 63% and 15% greater in isolated bladders from 29-month and 17-month animals, respectively, as compared to 7-month animals. The amounts of [3H]quinuclidinyl benzilate bound to membrane preparations were 46% and 7% greater. In contrast, no age-related changes were observed in phenylephrine-induced contraction or in isoproterenol-induced relaxation of bladder. Thus, the urinary bladder of aged rats appears to develop increased sensitivity to cholinergic stimuli because of an increase in the number of muscarinic cholinergic receptors.

Effect of long-term caloric restriction on brain monoamines in aging male and female Fischer 344 rats.

The present study examines the changes in central monoamines and their metabolites in aged male and female rats after long-term caloric restriction. Fischer 344 rats of both sexes (n = 5-10/group) were maintained on one of two dietary regimens: ad libitum NIH 31 diet or 60% by weight of the ad lib. intake (restricted), supplemented with vitamins and minerals. Animals received these diets from the age of 14 weeks until killed at 22.25 months of age. Caudate nucleus (CN), hypothalamus (HYPO), olfactory bulb (OB) and nucleus accumbens (NA) were assayed for content of norepinephrine (NE), dopamine (DA) and its metabolites (dihydroxyphenylacetic acid, DOPAC, and homovanillic acid, HVA) and serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) using HPLC/EC. Relative to the ad lib. group, restricted rats of both sex showed significant decreases in NE content in CN, HYPO and OB. DA and 5-HT content were decreased significantly in the CN and HYPO. No significant changes were found in the levels of DA metabolites in all brain regions studied. While the 5-HIAA level was significantly reduced in the HYPO and NA of the female restricted rats, it was increased several-fold in the OB of the male restricted animals. These preliminary results suggest that long-term caloric restriction alters brain monoamine concentrations, an effect which may in turn modify the normal rate of aging.

Effect of pretreatment with amphetamine on the interaction between amphetamine and dopamine neurons in the nucleus accumbens.

The present study was designed to examine the effect of pretreatment with amphetamine on the ability of amphetamine to release dopamine from slices of the nucleus accumbens and striatum and to stimulate locomotor activity or stereotyped behavior, after direct injection into either the nucleus accumbens or the striatum. Rats were injected twice daily for 5 days with either amphetamine (5 mg/kg, i.p.) or saline. At 33 days after this pretreatment, the release of endogenous dopamine from both regions of the brain in vitro by amphetamine and the changes in behavioral responses to the direct injection of amphetamine into either region were examined. Amphetamine at both 1 and 10 microM stimulated the release of endogenous dopamine from slices prepared from both of the brain areas. The release of dopamine by amphetamine was increased in rats pretreated with amphetamine. Consistent with its ability to stimulate endogenous release of dopamine, amphetamine, when injected into the nucleus accumbens, stimulated locomotor activity, while stereotyped behavior was enhanced when amphetamine was injected into the striatum. However, the locomotor activity and stereotyped behavioral responses to small doses of amphetamine (5, 10 or 25 micrograms) were not significantly greater in amphetamine-pretreated rats, compared to saline-pretreated animals. A greater stimulation of both responses in amphetamine-pretreated rats was only observed when a large dose (50 micrograms) of amphetamine was administered into either the nucleus accumbens or striatum.(ABSTRACT TRUNCATED AT 250 WORDS)

Time course of the development of the enhanced behavioral and biochemical responses to amphetamine after pretreatment with amphetamine.

These experiments were designed to examine the time course of development of the enhanced stereotyped behavioral response to amphetamine after withdrawal from chronic pretreatment with amphetamine and to determine whether this time course correlates with that of the enhancement in the amphetamine-induced stimulation of the release of dopamine (DA) from striatal slices. Rats were pretreated with amphetamine (5 mg/kg, i.p.) or saline, twice daily for 5 consecutive days. At 3, 15 and 30 days after withdrawal of the drug the stereotyped behavioral response and the release of endogenous DA from slices of striatum in response to a challenge dose of amphetamine were measured. At all 3 times tested, the stereotyped behavioral response to the challenge dose of amphetamine was enhanced in the rats pretreated with amphetamine, with the greatest degree of enhancement seen at 15 and 30 days after withdrawal of the drug. At these times, the responses were associated with a significant attenuation in the stimulation of locomotor activity produced by the challenge dose of amphetamine, which was probably related to the enhanced stereotyped behavioral response. Amphetamine stimulated the release of endogenous DA from slices of striatum in rats pretreated with saline and amphetamine. However, the release of endogenous DA from slices of rats pretreated with amphetamine was significantly greater than that of saline-pretreated rats at 15 and 30 days after withdrawal of the drug, but not at 3 days after withdrawal. Thus, pretreatment with amphetamine resulted in enhanced behavioral and biochemical responses to amphetamine which increased over time after withdrawal of the drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-related change in alpha-adrenergic responsiveness of the urinary bladder of the rat is regionally specific.

The effects of age on the responsiveness of the body of the urinary bladder and base of the bladder to alpha-adrenergic agonists were studied. Regions of the bladder were isolated from Fischer 344 rats, ages 7, 16, and 27 months. Maximum isotonic contractions elicited by potassium chloride (KCl) in both regions of the bladder were unaffected by age. In the bladder body there was an age-related increase in the maximum contraction elicited by phenylephrine, norepinephrine and clonidine. No such alteration in responsiveness was observed in the base of the bladder with age. The ED50 values of all three agonists were unchanged with age in both regions of the bladder. The pA2 values of prazosin and yohimbine were approximately 8.5 and 6.0, respectively, in the body of the bladder, and these values were not altered by age. Thus, it is concluded that an age-related increase occurs in the responsiveness of the body of the bladder to alpha-adrenergic activation and that these changes are mediated by alpha 1-adrenoceptors.

Ontogeny of the enhanced behavioral response to amphetamine in amphetamine-pretreated rats.

Repeated administration of amphetamine to adult rats results in enhanced behavioral responses to subsequent amphetamine exposure. These experiments were designed to determine the earliest age at which behavioral sensitization to amphetamine could be detected. Rats from both sexes (n = 6-8/group) at ages of 1, 7, 21 or 49 postnatal days (PNDs) were injected with either d-amphetamine sulfate (5 mg/kg) or saline, SC, twice daily for 5 consecutive days. Stereotyped behavior and locomotor activity responses to a challenge dose of d-amphetamine (2.5 mg/kg), or saline, IP, were assessed for a total of 90 min, 15 days after the last dose of pretreatment. Amphetamine-induced stereotyped behavior was significantly enhanced only when amphetamine pretreatment was initiated at PND 49, but not at the earlier ages of PND 1, 7 or 21. There was no apparent sex difference in this effect. Correspondingly, amphetamine-induced locomotor activity was reduced in both sexes of the same age group (PND 49), but not in groups pretreated earlier, when compared to the saline-pretreated rats. These results suggest that amphetamine sensitization may be a late-developing effect, one which occurs sometime after the 3rd week of postnatal life.

Prenatal cocaine exposure affects postnatal dopaminergic systems in various regions of the rat brain.

Pregnant female Sprague-Dawley rats were injected once daily with either 40 mg/kg cocaine hydrochloride or 0.9% saline from gestation day (GD)12 to GD 21. On postnatal day (PND)30, male offspring were sacrificed and fresh tissue from the striatum (ST) and nucleus accumbens (NA) was dissected for assessment of dopamine (DA) receptor affinity, DA uptake and DA release. 10(-6) M cocaine inhibited [3H]-DA uptake in ST tissue, whereas 10(-4) and 10(-5) M cocaine inhibited [3H]-DA uptake in the NA tissue of postnatally exposed cocaine offspring verses saline-treated controls (p < 0.05). DA release stimulated by 10(-6) M amphetamine was significantly reduced in both the ST (p < 0.001) and NA (p < 0.01) of postnatal offspring exposed to cocaine in utero compared with saline controls. In utero cocaine exposure did not influence offspring ST or NA dopamine 1 (D1) dissociation constant (Kd) or receptor density (Bmax). However, the treatment group experienced a significant increase of binding affinity for the ST D2 receptor with no change in D2 Bmax. The treatment group also experienced no change in D2 receptor binding affinity or number of binding sites in the NA. These results show that in utero exposure to cocaine results in altered postnatal dopaminergic function.

Behavioral responses to repeated cocaine exposure in mice selectively bred for differential sensitivity to pentobarbital.

Mice from the 20th generation of three lines divergently selected for response to pentobarbital-induced sedation times [long-sedation time (LST), short sedation time (SST), and randomly bred control (RBC)] were used to study cocaine-induced behavioral sensitization. These lines showed variable degrees of locomotor activities in response to cocaine. At a low cocaine dose and long withdrawal period (10 mg/kg, twice a day for 5 days followed by a 14-day withdrawal), the LST mice showed tolerance development. In response to cocaine, the locomotor activities of the SST were not significantly different from the RBC group. At a higher dose and a shorter withdrawal period (20 mg/kg, daily for 7 days followed by a 3-day withdrawal), the SST mice showed behavioral sensitization similar to the RBC mice, but the LST mice did not develop sensitization. The different responses in locomotor activity induced by cocaine suggest that genetic factors may play a role in determining the magnitude of response to this drug. Dopamine (DA) levels did not differ significantly in either striatum (STR) or nucleus accumbens (NAC) for the cocaine-treated animals to their corresponding saline-treated controls. The affinity (Kd) of D2 in the NAC decreased significantly, without changes in density (Bmax), in the cocaine-treated SST and RBC mice. On the other hand, the density of D2 binding sites in the SST and the RBC mice in the STR was significantly increased in cocaine-treated groups without change in Kd. The LST mice did not show any changes in the Kd and Bmax in either the STR or the NAC. Taken together, these findings suggest that the changes in the Kd of D2 in the NAC and the Bmax of D2 in the STR may contribute to the differences in locomotor responses to cocaine exposure in these mouse lines.

Repeated administration of cocaine alters dopamine uptake and release in the striatum nucleus accumbens.

Male Sprague-Dawley rats were administered 25 mg/kg, intraperitoneally (i.p.) cocaine-HCI twice daily for 14 consecutive days (total of 50 mg/kg), while control animals received an equivalent volume of 0.9% saline. After three days of withdrawal, the animals were sacrificed for dissection of striatal (STR) and nucleus accumbens (NA) brain regions. The treated group demonstrated a dose-dependent reduction for in vitro cocaine inhibition of [3H]dopamine (DA) uptake in the NA tissue verses controls. There were no significant differences amongst the treated and control groups for in vitro cocaine inhibition of [3H]DA in the STR. In vitro d-amphetamine (1, 5 and 10 microM)-stimulated DA release from STR tissue was not significantly different between the treated and the control groups. However, there was a significant decline in basal STR DA release and a significant enhancement of d-amphetamine (1 and 5 microM)-stimulated DA release in the NA for the treatment group versus controls. The results from the present study indicates sensitization to cocaine is primarily related to DA uptake and release in the NA.

Endogenous opioid peptide mediation of hypoalgesic response in long-term diabetic rats.

This study was designed to examine the role of endogenous opioid peptide mediation of elevated pain threshold in adult male Sprague-Dawley rats with long-term diabetes mellitus induced by streptozotocin (STZ). Diabetes resulted in a significant elevation in pain threshold as measured by the tail-flick and/or hotplate latency tests. The hypoalgesic response in diabetic rats to hotplate testing developed gradually over a 4-6 week period after a transient hyperalgesia during the first two weeks of diabetes. The elevation of pain threshold achieved peak level by the fourth week after STZ administration, and remained at that level throughout the experimental period (up to 13 weeks). This hypoalgesic state in diabetic animals is thought to be mediated by opioid receptors (i.e. mu and delta). The involvement of the mu receptor is supported by the effect of naltrexone on the STZ-diabetic rats; naltrexone significantly attenuated the increase in tail-flick and hotplate latencies, compared to that of the non-diabetic controls. Furthermore, the concentration of native (free) Met-enkephalin in the spinal cord of STZ-diabetic rats was about 5-fold higher than that of non-diabetic animals. Such high levels of Met-enkephalin suggest the involvement of delta opioid receptors in the hypoalgesic response observed in STZ-diabetic rats. Seven weeks of insulin treatment, initiated after development of the hypoalgesic response, normalized not only plasma glucose level and body weight of diabetic rats, but also returned their antinociceptive latency toward normal. The results of this study showed that long-term diabetes is associated with altered pain threshold and further support the hypothesis for endogenous opioid peptide mediation of hypoalgesia in chronically diabetic rats that can be prevented by insulin treatment.

Effect of diabetes on the levels of two forms of Met-enkephalin in plasma and peripheral tissues of the rat.

Levels of native and cryptic or peptidase-derivable (after being digested with trypsin and carboxypeptidase) Met-enkephalin were measured by a specific radioimmunoassay method in plasma, anterior and neurointermediate lobes of pituitary and various peripheral tissues of streptozotocin (STZ) diabetic rats. The results show that the highest concentration of native and cryptic Met-enkephalin were found in the neurointermediate lobe of pituitary. Streptozotocine-induced diabetes alters the concentration of either or both forms of Met-enkephalin in plasma, the anterior and neurointermediate lobes of the pituitary, heart, lung, spleen, liver, seminal vesicle, vas deferens, kidney, bladder detrusor, and duodenum. One of the most pronounced effects of diabetes observed in this study is seen in the seminal vesicles where native Met-enkephalin was depleted to less than 10% of the control value. The uneven distribution of Met-enkephalin in peripheral tissues may suggest that these tissues process and/or metabolize Met-enkephalin to different degrees. Our data also suggest that STZ-induced diabetes alters the enkephalinergic activity in some of these tissues. It is suggested that some of the peripheral pathophysiological symptoms associated with diabetes may be attributed, in part, to altered activity of enkephalinergic systems.

Effect of methylphenidate pretreatment on the behavioral and biochemical responses to amphetamine.

After rats were chronically pretreated with methylphenidate (MP), the stereotyped behavioral response to amphetamine (AMPH) was significantly enhanced. In addition, after this pretreatment, the AMPH-induced release of endogenous dopamine from slices of striatum and nucleus accumbens was also enhanced. These effects of MP pretreatment are similar to those reported for AMPH pretreatment and suggest that the sensitization to AMPH that develops after pretreatment with either drug is due to a common mechanism.

Dietary choline affects response to acetylcholine by isolated urinary bladder.

Age-related increases occur in the response of isolated urinary bladders to the parasympathetic neurotransmitter acetylcholine (ACh). Experiments were carried out to determine whether long-term elevation or diminution in the amount of ingested choline can also affect the response of the urinary bladder to ACh. Female C57BL/6J mice were maintained on a choline-deficient chow and on drinking water supplemented with either 0, 1.5, or 4.0 mg/ml choline chloride from 8 to 20 months of age. Isolated bladders from choline deficient animals showed a 46% increase in the maximum response to ACh as compared to those from normal choline animals, while bladders from animals on choline enriched diets showed a 15% decrease in maximum contractile response. Radioligand binding experiments suggested that the functional changes result from alterations in the density of muscarinic receptors in the bladder. The results are consistent with the hypothesis that muscarinic receptors are down-regulated to compensate for increased parasympathetic activity associated with choline-enriched diets and up-regulated to compensate for decreased parasympathetic activity associated with choline-deficient diets.

Effects of time of day on chlorpyrifos-induced alterations in body temperature.

Chlorpyrifos (CPF) is an organophosphate (OP) insecticide and is among the most common and widely used commercial insecticides. Human intoxication is reported to result in a typical set of responses, which include an immediate and long lasting hyperthermic response (fever). Rodents exposed to similar doses exhibit a biphasic body temperature response: short-term hypothermia followed by subtle hyperthermia several days after administration. Time of day of administration has been suggested to alter the body temperature effect of CPF. In the present study, it is shown that adult male Sprague-Dawley rats exposed to CPF via (oral) gavage at four different times of the day demonstrate a hypothermic response, the timing and magnitude of which is independent of time of exposure and that is blocked by atropine pretreatment. However, a delayed (hyperthermic) response seems to be exhibited only when dosing occurs during the light phase. Our findings support existing theories that the hypothermic and hyperthermic effects of CPF work through independent mechanisms. It is also suggested that humans may indeed exhibit a biphasic temperature response to CPF intoxication, but that it is not typically detected.

Behavioral and neurochemical effects of orally administered MDMA in the rodent and nonhuman primate.

MDMA (methylenedioxymethamphetamine) is a recreational drug of abuse known as "Ecstasy" which markedly decreases regional brain serotonin (5-HT) content and produces 5-HT nerve terminal degeneration in forebrain areas of the rat. In order to determine the acute and chronic behavioral effects of MDMA, adult rats were given MDMA at 0, 5 or 10 mg/kg, po for 4 consecutive days. Alternatively, parachloroamphetamine (PCA) at 5 mg/kg was administered under the same regimen. Within 30 min after the first dose, the MDMA-treated rats exhibited the serotonin motor syndrome consisting of straub tail and splayed hindlimbs comparable to that seen in the PCA-treated rats. This serotonin motor syndrome, with a duration of about 2 hr, was less pronounced after subsequent doses. At 2-4 wk after the last dose, no significant differences between control and treated rats were seen in emergence, hot plate response, auditory startle response or complex maze behavior even though a significant dose-related decrease (50%) in 5-HT concentration was observed in the frontal cortex and hippocampus of these rats 4 wks after the last dose. Adult female monkeys dosed po with 5 or 10 mg/kg of MDMA twice/day for 4 consecutive days demonstrated no spontaneous behavioral changes or weight loss compared to controls, but forebrain 5-HT concentration was reduced by 80% 1 mon after dosing. These data indicate that at doses only 2-3 times the human dose, MDMA produces significant forebrain 5-HT decreases but does not produce detectable residual behavioral alterations as assessed by these behavioral paradigms.

The role of inducible nitric oxide synthase in cocaine-induced locomotor sensitization.

Experimentally naive male Sprague-Dawley rats (weighing 85-110 g) were used to examine the role of inducible nitric oxide synthase (iNOS) in cocaine-induced locomotor sensitization. Repeated administration of cocaine (15 mg/kg, ip) for 7 consecutive days produced locomotor sensitization. Pretreatment with iNOS inhibitors, L-N(6)-(1-iminoethyl) lysine (NIL) or (-)-epigallocatechin gallate (EGCG; 10 mg/kg, ip), 30 min before cocaine (15 mg/kg, ip) administration totally blocked the development of cocaine-induced locomotor sensitization. Dopamine (DA) receptor binding in the nucleus accumbens (NAC) showed a significant decrease in the density of D(2) receptor and the affinity of D(1) receptor after cocaine treatment. Pretreatment with EGCG or NIL abolished the cocaine-induced changes in these parameters. These results suggest that iNOS may participate in the process of development of locomotor sensitization through the modulation of DA receptors in the NAC.

Opioid modulation of amphetamine-stimulated dopamine release and concentration in rat striatal slices.

The effects of morphine and naltrexone on amphetamine-stimulated release and total concentration of dopamine (DA) from rat striatal slices in vitro were examined in this study. Adult male Sprague-Dawley rats were sacrificed and the striata were dissected, sliced, and then incubated in buffer solution at 37 degrees C with amphetamine in the presence or absence of various concentrations of morphine, naltrexone (or both), or the dihydroxyphenylalanine (DOPA) decarboxylase inhibitor (NSD-1015). The concentrations of DOPA, DA, and dihydroxyphenylacetic acid (DOPAC) in the tissue slices and buffer media were measured by HPLC/EC. Amphetamine enhanced DA release and also increased total DA concentrations. However, neither morphine nor naltrexone alone altered DA concentration in the media or tissue slices relative to control (no drug added). Moreover, neither morphine nor naltrexone at 1, 10, or 100 microM altered amphetamine-stimulated DA release. However, morphine (1 or 10 microM) inhibited the amphetamine-stimulated increase in total concentration of DA. This effect of morphine was blocked by naltrexone. NSD-1015 alone or in combination with morphine did not alter amphetamine-stimulated DA release, but significantly reduced DA concentration in striatal slices. NSD-1015 alone also increased DOPA accumulation in both media and tissue slices, and this effect was inhibited by the addition of morphine. These results indicate that morphine inhibits the amphetamine-stimulated increase in total DA content, but not the amphetamine-stimulated release of DA.

Hypothermic effect of ethanol in mice selected for differential sleep-time response to pentobarbital.

The hypothermic action of ethanol was investigated in genetically distinct lines of mice selected for sleep-time response to pentobarbital for six generations. Ethanol (3 g/kg, intraperitoneally) was administered to alcohol-naive males and females from each of the unselected control, long-, and short-sleep mouse lines. Rectal temperatures were measured immediately before, and at 15, 30, 60, 90, 120, and 240 min after ethanol injection. Eight female and eight male mice from each line were sacrificed at each time point, and trunk blood was collected for plasma ethanol analysis. The results show that short-sleep mice were less hypothermic (p < 0.05) compared to long-sleep mice at 15 and 30 min after ethanol administration. However, plasma ethanol concentrations were not significantly different between the mouse lines at any time point. Therefore, the line-dependent differential ethanol-induced hypothermia observed may be a result of differences in "brain sensitivity" rather than in the rates of ethanol metabolism among the mouse lines.