Mouse Strain Differences in Oral Operant Ethanol Reinforcement under Continuous Access Conditions

The present experiment examined ethanol setf-administration in C57BL/6J (C57) and DBA/2J (DBA) mice using a continuous access operant procedure. Adult male C57 and DBA mice were initially trained to perform a lever press response to obtain access to 10% w/v sucrose solution. Subsequently, the mice were placed in operant chambers on a continuous (23 hr/day) basis with access to food (FR1), 10% v/v ethanol (FR4), and water from a sipper tube. C57 mice displayed greater rates of responding on the ethanol-associated lever compared with DBA mice. Responding on the food lever was the same in both strains, but DBA mice consumed greater amounts of water. C57 mice consistently displayed both prandial and nonprandial episodes (bouts) of ethanol responding. DBA mice did not respond for ethanol in bouts. Following 50 consecutive sessions, ethanol concentration was altered every 5 days. Response patterns were determined using 0, 5, 10, 20, and 30% v/v ethanol concentrations. C57 mice displayed concentration-dependent responding on the ethanol lever showing that ethanol was functioning as an effective reinforcer in this strain. In contrast, responding on the ethanol lever by DBA mice did not change as a function of ethanol concentration. Saccharin (0.2% w/v) was subsequently added to the ethanol mixture, and responding was examined at 0, 5, 10, and 20% ethanol concentrations. Overall, ethanol lever responding was increased in both strains. As before, C57 mice showed higher levels of ethanol responding, compared with DBA mice. C57 mice also showed higher responding for saccharin alone. These results are consistent with findings that suggest orally administered ethanol is a more effective reinforcer in C57 mice than in DBA mice. Furthermore, C57 mice engage in ethanol-reinforced responding over a broader range of conditions than DBA mice.     

Tolerance to Ethanol Hypothermia in Inbred Mice: Genotypic Correlations with Behavioral Responses

Hypothermia was studied 5 min before, and 30 and 60 min after intraperitoneal administration of ethanol (3 g/kg) in 20 inbred strains of mice. Ethanol was given daily for 8 days, and temperatures were taken on Days 1, 3, 5, and 8. Tolerance was indexed by the reduction in hypothermia over days. There were large strain differences in baseline temperature, the hypothermic effect of ethanol, and in development of tolerance to hypothermia. Some strains of mice (DBA/1J, DBA/2N, MA/MyJ, and PL/J) did not develop tolerance to the hypothermic effect of ethanol. Initial sensitivity to the hypothermic effect of ethanol was significantly genetically correlated with tolerance development, indicating control of these responses by common genes. Ethanol-induced changes in activity and ataxia, as well as blood ethanol concentrations, were also assessed. Although there were significant strain differences in activity reduction, ataxia, blood-ethanol concentrations, and changes in these parameters during the course of chronic treatment, none of these variables could explain the genetic differences in hypothermic sensitivity and tolerance.     

Withdrawal Severity After Chronic Intermittent Ethanol in Inbred Mouse Strains

Background: To study withdrawal, ethanol is usually administered chronically without interruption. However, interest has recurred in models of episodic exposure. Increasing evidence suggests that chronic intermittent exposure to ethanol leads to a sensitization effect in both withdrawal severity and ethanol consumption. The goal of the present study was to examine mouse inbred strain differences in withdrawal severity following chronic intermittent exposure using the handling‐induced convulsion as the behavioral endpoint. We also sought to compare the withdrawal responses of inbred strains across acute, chronic continuous, and chronic intermittent exposure regimens. Methods: Male mice from 15 standard inbred strains were exposed to ethanol vapor for 16 hours each day for 3 days and removed to an air chamber during the intervening 8 hours. Mice in the control groups were handled the same, except that they were exposed only to air. Daily blood ethanol concentrations were averaged for each mouse to estimate total dose of ethanol experienced. Results: Across strains, mice had an average daily blood ethanol concentration (BEC) of 1.45 ± 0.02 mg/ml and we restricted the range of this value to 1.00–2.00 mg/ml. To evaluate strain differences, we divided data into two dose groups based on BEC, low dose (1.29 ± 0.1 mg/ml) and high dose (1.71 ± 0.02 mg/ml). After the third inhalation exposure, ethanol‐exposed and air‐exposed groups were tested hourly for handling‐induced convulsions for 10 hour and at hour 24 and 25. Strains differed markedly in the severity of withdrawal (after subtraction of air control values) in both dose groups. Conclusion: The chronic intermittent exposure paradigm is sufficient to elicit differential withdrawal responses across nearly all strains. Data from the high‐dose groups correlated well with withdrawal data derived from prior acute (single high dose) and chronic continuous (for 72 hours) ethanol withdrawal studies, supporting the influence of common genes on all three responses.     

Comparative Pharmacokinetics of Ethanol in Inbred Strains of Mice Using Doses Based on Total Body Water

The mean total body water was determined by desiccation in DBA/2J, CBA/J, and C57BL/6J mice to be 60.6, 65.6, and 68.6 percent of body weight, respectively. The pharmacokinetics of ethanol was subsequently studied in mice of these strains given an intraperitoneal dose of 116 mmoles/l of total body water based on the desiccation study. This dose was equivalent to 70, 76, and 80 mmoles/kg in the DBA/2J, CBA/J, and C57BL/6J strains, respectively. The zero time concentrations were nearly identical between strains; therefore volume of distribution (VD) estimates based on mmole/kg doses reflected interstrain differences in total body water. The apparent zero order elimination rate was significantly greater in the DBA/2J strain versus the other two strains using this regimen. Interstrain differences in ethanol sleep time paralleled the differences in anesthetic sensitivity evidenced by blood concentrations at the time of regaining the righting reflex. The results demonstrate the importance of considering differences in total body water and hence ethanol VD when comparing the effects of ethanol in inbred mouse strains.     

Intravenous Ethanol Self-administration in C57BL/6J and DBA/2J Mice

Two strains of mice, C57BL/6J (B6) and DBA/2J (D2) were allowed to self-administer intravenous (iv) ethanol. These two strains were selected because they differ greatly in their preference for drinking ethanol solutions: 86 mice are preferrers, whereas D2 mice are avoiders of ethanol. Of interest was whether these strains would also differ in self-administration of iv ethanol when taste factors presumably do not influence consumption. Mice were trained with either 60, 75, or 90 mg/kg per infusion. Mice from both strains acquired nosepoking for all of these doses on an FR-3 schedule of reinforcement during 2-hr daily sessions. Additionally, mice in both strains acquired an equal preference for nosepoking on the side resulting in ethanol infusions, compared with the side that had no scheduled consequence, although B6 mice took somewhat more ethanol early in training than did D2 mice. Mice in both strains achieved equal levels of responding at the conclusion of training, when response rates had stabilized. A subset of animals were then tested at doses of ethanol ranging from 25 to 125 mg/kg per infusion. Although their responding tended to decrease over time regardless of changes in the unit dose of ethanol, these mice showed lower response rates for higher doses of ethanol, and less responding for saline than for ethanol. Together, these findings imply that iv ethanol has reinforcing properties in both these strains, despite the strain difference in preference for oral ethanol. Self-administration of iv ethanol in mice may prove a valuable addition to existing animal models for the study of ethanol reward.     

Acute alcohol withdrawal is associated with c-Fos expression in the basal ganglia and associated circuitry: C57BL/6J and DBA/2J inbred mouse strain analyses

BACKGROUND: The DBA/2J (D2) and C57BL/6J (B6) mouse strains are the most widely studied genetic models of severe and mild acute alcohol withdrawal, respectively. Previous studies have identified quantitative trait loci and genes involved in risk for acute ethanol withdrawal using mapping populations derived from the D2 and B6 strains, but the brain region(s) and circuit(s) by which these genes and their protein products influence ethanol physiological dependence and associated withdrawal remain to be elucidated. METHODS: B6 and D2 were administered a sedative-hypnotic dose of ethanol (4 g/kg) or saline (control) and returned to their home cages where they were left undisturbed for 7 hr, which has been shown in previous studies to correspond to peak acute ethanol withdrawal severity. The mice were then euthanized and assessed for their numbers of c-Fos immunoreactive neurons across 26 brain regions. The question addressed was whether or not ethanol-withdrawn D2 and B6 mice differed in c-Fos induction (neural activation) within circuitry that could explain the severe ethanol withdrawal of the D2 strain and the mild ethanol withdrawal in B6 strain mice. RESULTS: At peak acute ethanol-withdrawal ethanol-withdrawn D2 and B6 mice differed in neural activation within the basal ganglia, including the subthalamic nucleus and the two major output nuclei of the basal ganglia (the medial globus pallidus and the substantia nigra pars reticulata). Genotype-dependent c-Fos induction was also apparent in associated circuitry including the lateral septum, the ventral tegmental area, the nucleus accumbens core, the dorsolateral caudate putamen, the substantia nigra pars compacta, the cingulate and entorhinal cortices, and the ventral pallidum. D2 and B6 mice showed comparable neural activation in the bed nucleus of the stria terminalis, and the nucleus accumbens shell. CONCLUSIONS: The present studies are the first to use immediate early gene product expression to assess the pattern of neural activation associated with acute ethanol withdrawal. Our results point to the involvement of an extended basal ganglia circuit in genetically determined differences in acute ethanol withdrawal. Based on these data, we suggest that quantitative trait genes (QTGs) involved in acute ethanol withdrawal exert their effects on this phenotype via one or more of the brain regions and circuits identified. As more information becomes available that integrates neural circuit and QTG analyses, the precise mechanisms by which QTGs affect ethanol physiological dependence and associated withdrawal will become apparent.     

GOLD KINETICS UNDER LONG-TERM TREATMENT WITH GOLD(I) DISODIUM THIOMALATE: A COMPARISON IN THREE DIFFERENT MOUSE STRAINS

Following weekly i.m. injections of gold(I) disodium thiomalate(GST), mice of strains A.SW and C57BL/6 develop adverse immunereactions, whereas DBA/2 mice do not. We have studied the pharmaco-toxicokineticsof gold in these strains under chronic GST treatment. Our resultsindicate that the susceptible strains A.SW and C57BL/6 accumulatesignificantly higher gold concentrations in the liver and spleencompared to the resistant strain DBA/2. In the kidney of DBA/2mice, gold concentrations persisted at a plateau level, whereasin A.SW and, particularly, C57BL/6 mice early peaks of goldconcentrations were followed by a transient decrease, suggestiveof tubular toxicity. Whereas splenic T and B cells failed tocontain measurable gold concentrations in all three strains,splenic and peritoneal macrophages contained relatively highlevels, more so in the susceptible strain C57BL/6 than in theresistant DBA/2 strain. This finding is consistent with theconcept that macrophages play an important role in both theadverse and the beneficial effects of gold drugs. KEY WORDS: Anti-rheumatic drugs, Gold sodium thiomalate, Genetics, Inbred mouse strains, Macrophages, Pharmacokinetics, Toxicokinetics.     

The effect of dietary manipulation on hepatic lipid accumulation in rats undergoing small intestinal bypass

Small intestinal bypass performed for morbid obesity produces hepatic fat infiltration which persists in some patients for more than 5 years. In an attempt to define causative and preventative factors of hepatic steatosis following small intestinal bypass, a rat model was developed. In this study, nutritionally obese rats underwent sham operations or bypass of 90 per cent of their small intestine and postoperatively were fed various diets to determine the effects of dietary manipulations on hepatic lipid content. After 30 days the rats were killed and their hepatic lipid content and lipogenic enzyme activity determined. In rats that underwent intestinal bypass neither a high fat, a high carbohydrate nor a high protein diet increased hepatic lipid content over that present in sham operated animals. A low (4.5 per cent) protein diet increased total hepatic lipid and hepatic triglyceride content. The increased triglyceride levels were not associated with significant changes in lipogenic enzyme activity and were associated with decreased serum triglycerides suggesting impaired triglyceride transport from the liver secondary to decreased lipoprotein formation as a possible etiologic mechanism. A significant inverse relationship was found between hepatic triglyceride content and hepatic protein content. These results support previous reports from human studies of hypoproteinemia associated with hepatic steatosis following small intestinal bypass.     

The effects of moderate ethanol consumption on the liver of the monkey, Macaca fascicularis

BACKGROUND: Although evidence has accumulated for the cardioprotective effects of moderate ethanol consumption, little is known about the effects on the liver of consuming the equivalent of two drinks per day. The objective of this study was to determine the effects of moderate ethanol administration on the hepatic content of enzymes involved in ethanol oxidation, on hepatic lipid accumulation, and on serum markers of liver function/damage in the monkey, Macaca fascicularis. METHODS: Ovariectomized, adult monkeys were maintained for 34 months on an atherogenic diet containing cholesterol 1.21 mg/kJ. They were trained to drink ethanol plus vehicle at a dose of 0.5 g/kg body weight, which was administered 5 days a week for 2 years. Blood was collected for ethanol concentrations (1 hr after ethanol administration) and was also assayed for gamma-glutamyltransferase, alanine aminotransferase (ALT), and alkaline phosphatase (ALP) activities. Liver obtained at necropsy was analyzed for triglyceride and cholesterol contents and for alcohol dehydrogenase, cytochrome P450 2E1, and cytochrome P450 3A4 by Western blots. RESULTS: The blood ethanol concentrations measured 1 hr after ethanol administration were relatively constant over the 2-year dosing period. Hepatic levels of alcohol dehydrogenase and the cytochrome P450s were not significantly different between ethanol-consuming animals and control animals. Ethanol-associated increases in liver triglyceride were not significant due to high variability in hepatic lipid content in both the controls and ethanol consumers. However, covariance analyses using pretreatment concentrations of plasma cholesterol and apolipoprotein A-I suggested that the ethanol-related increase in hepatic free cholesterol was significant. Relative to controls, alcohol consumers had higher levels of serum ALT and a transient increase in ALP at 5 months. CONCLUSIONS: The observations made in this study on primates administered an atherogenic diet suggest that moderate ethanol ingestion has modest effects on the liver, including slightly increased ALT and ALP values. However, additional studies will be required to verify that this level of consumption is hepatotoxic when ingested over extended periods. This is still a concern because some human studies suggest that levels of ethanol considered to be cardioprotective cause liver injury when consumed over a lifetime.     

Alterations of hepatic microsomal enzymes in the early phase of murine schistosomiasis

Schistosoma mansoni has been reported to cause a downregulation of hepatic cytochrome P450 activities after granulomas are formed around worm eggs harbored in the mouse liver. Only a few studies, however, provided data on the activity of xenobiotic-biotransaformation enzymes in the early phase of S. mansoni infection. In this study, we evaluated the alterations of liver microsomal enzymes during early infection (post-infection days, PIDs, 15 and 30) when granulomas are not found in the mouse liver yet. Swiss Webster (SW) and DBA/2 mice of either sex were infected with 100 S. mansoni cercariae on postnatal day 10. Levels of total-CYPs and activities of alkoxyresorufin-O-dealkylases (EROD, MROD, PROD and BROD), N-nitrosodimethylamine-N-demethylase (NDMA-d), coumarin 7-hydroxylase (COH, DBA/2 only) and UDP-glucuronosyltransferase (UGT) were measured in liver microsomes from mice killed on PIDs 15 and 30. Age-matched (sham-infected) mice of the same sex and strain were used as controls. Neither total-CYP levels nor microsomal enzyme activities were altered in SW and DBA/2 mice on PID 15. On PID 30, total-CYP levels, and COH, PROD and UGT activities remained unaltered, while gender- and strain-specific minor changes of EROD, MROD, BROD and NDMA-d (i.e., increase in SW and reduction in DBA/2) were found. In conclusion, our results suggest that, contrasting to a consistent and almost generalized downregulation of CYPs in chronic schistosomiasis, alterations of hepatic CYPs in early (acute) infection are isoform and mouse's gender and strain specific.     

Dopaminergic neurons in the ventral tegmental area of C57BL/6J and DBA/2J mice differ in sensitivity to ethanol excitation

BACKGROUND: The mesolimbic dopamine pathway that originates in the ventral tegmental area (VTA) is important for the rewarding effects of ethanol. Ethanol has been shown to excite dopaminergic neurons of the VTA, both in vivo and in vitro, in rats. Behavioral differences in the rewarding effects of ethanol have been observed between C57BL/6J and DBA/2J mice. The present electrophysiological study examined the effect of ethanol on individual dopaminergic VTA neurons from these two inbred mouse strains. METHODS: Extracellular single unit recordings of spontaneous action potentials were made from dopaminergic VTA neurons in brain slices from either C57BL/6J or DBA/2J mice. Ethanol (10 to 160 mM) was administered in the superfusate and the mean change in firing rate produced by ethanol was measured. RESULTS: There was no significant difference in basal spontaneous firing rate of dopaminergic VTA neurons between these two mouse strains. Ethanol caused a concentration-dependent increase in the firing rate of neurons from both mouse strains. Ethanol excited dopaminergic VTA neurons from DBA/2J mice more potently than those from C57BL/6J mice. CONCLUSIONS: The difference in sensitivity to ethanol excitation of dopaminergic VTA neurons in C57BL/6J and DBA/2J mice may contribute to differences in their behavioral response to ethanol. The fact that a given concentration of ethanol causes greater excitation of dopaminergic VTA (reward) neurons in DBA/2J mice than in C57BL/6J mice could explain why DBA/2J mice show much stronger place preference conditioning with ethanol. The higher voluntary intake of ethanol by C57BL/6J mice may be partly due to the insensitivity of their dopaminergic VTA neurons that requires them to drink a lot of ethanol to achieve sufficient excitation of reward neurons, whereas DBA/2J mice avoid oral ingestion of ethanol, despite its rewarding effect, because of their aversion to its taste.     

Blockade of the leptin-sensitive pathway markedly reduces alcohol consumption in mice

BACKGROUND: The neuropeptide leptin links adipose stores with hypothalamic centers and serves as an endocrine signal involved in the regulation of appetite (and possibly in the endorphinergic modulation of the drug reward system). Increased plasma leptin has been observed at the onset of alcohol withdrawal in humans, and ethanol consumption after withdrawal was increased by injection of leptin in mice. We addressed the role of leptin in alcohol-related behaviors by studying ethanol consumption in two strains of spontaneously mutant mice that lack leptin (ob/ob) or the leptin receptor (db/db). METHODS: Two strains of mutant leptin-deficient (ob/ob) or leptin-resistant (db/db) mice were tested in a two-bottle-choice paradigm and were compared with wild-type (C57BL/6 inbred strain) mice. The effects of leptin injection on voluntary ethanol intake have been investigated in ob/ob and C57BL/6 mice. RESULTS: Males and females of both mutant strains showed a significantly lower preference for alcohol in a two-bottle-choice paradigm compared with wild-type mice. Male ob/ob mice demonstrated slightly higher avoidance of bitter taste, and females of the both mutant strains showed a reduced preference for saccharin solutions. Administration of leptin (1 mg/kg intraperitoneally, daily for 8 days) altered body weight but failed to increase the preference for ethanol in ob/ob mice; i.e., we could not correct the effects of leptin deficiency on alcohol consumption by the injection of leptin. Also, there were no differences between the effects of leptin (1 mg/kg intraperitoneally, daily for 8 days) and saline injections on alcohol consumption in C57BL/6 mice. CONCLUSIONS: These data show that blockade of the leptin pathway markedly decreases the preference for alcohol intake, but this decrease may be the result of compensatory or developmental changes in other systems rather than a more direct effect of leptin on alcohol consumption.     

Hepatic taurine concentration and dietary taurine as regulators of bile acid conjugation with taurine.

Taurine concentration in liver biopsies taken from 20 patients undergoing cholecystectomy or laparotomy for obstructive jaundice correlated with percentage of taurine-conjugated bile acids in hepatic bile. In biliary obstruction, taurine concentrations in muscle did not parallel high hepatic taurine concentrations, suggesting selective hepatic taurine accumulation in biliary obstruction. In fasting subjects with an intact bile acid enterohepatic circulation, per cent taurine-conjugated bile acids in bile was the same as per cent taurine conjugation of bile acids by the liver. Ingestion of small amounts of taurine (250 mg) can increase the per cent taurine conjugation by the liver. Of 10 subjects, 7 increased per cent taurine conjugation of bile acids by the liver by 2.5 to 10% at 2 1/2 hr after intraduodenal taurine administration. We conclude that hepatic taurine concentration is a major determinant of per cent taurine conjugation of bile acids by the liver in man. In the fasting subject with an intact enterohepatic circulation, per cent taurine-conjugated bile acid in the bile acid pool is very close to the per cent taurine conjugation of bile acids by the liver. Hepatic bile acid conjugation pattern may differ from that of the bile acid pool as a result of taurine ingested with meals, but the deviation is small, and acute alteration of the per cent taurine conjugation in the bile acid pool does not occur.     

Correlation of hyperplasia, splenomegaly and hepatomegaly with parasite population in BIO.LP-a mice infected with Leishmania donovani.

Congenic strain of BIO.LP-a male mice were experimentally infected with Leishmania donovani 3S strain from the spleen of a hamster donor. The weight ratio of spleen body, liver body and spleen liver calculated from the weight of six mice taken at weekly intervals for a period of 49 days showed that there is no hepatomegaly or splenomegaly during the first 14 days of infection when the parasite population increases. Maximum enlargement of liver and spleen was observed at day 35 postinfection, when the parasite population had declined to 11 per cent. Slight recovery was noted at day 49 with parasites reducing to 1 per cent. It is suggested that in endemic areas of human visceral leishmaniasis a search for amastigotes should be made from the biopsy of the liver or sternal puncture during the first phase of the disease when the patients suffer from a high fever with rigors. With the enlargement of liver and spleen due to proliferative response and infiltration of plasma cells, the parasite population disappears.     

Fatty liver induced by tetracycline in the rat. Dose-response relationships and effect of sex.

Dose-response relationships, biochemical mechanisms, and sex differences in the experimental fatty liver induced by tetracycline were studied in the intact rat and with the isolated perfused rat liver in vitro. In the intact male and female rat, no direct relationship was observed between dose of tetracycline and hepatic accumulation of triglyceride. With provision of adequate oleic acid as a substrate for the isolated perfused liver, a direct relationship was observed between dose of tetracycline and both accumulation of triglyceride in the liver and depression of output of triglyceride by livers from male and female rats. Marked differences were observed between female and male rats with regard to base line (control) hepatic concentration of triglyceride and output of triglyceride. Accumulation of hepatic triglyceride, as a per cent of control values, in response to graded doses of tetracycline, did not differ significantly between male, female and pregnant rat livers. However, livers from female, and especially pregnant female rats, were strikingly resistant to the effects of tetracycline on depression of output of triglyceride under these experimental conditions. These differences between the sexes could not be related to altered disposition of tetracycline or altered uptake of oleic acid. Depressed hepatic secretion of triglyceride accounted only for 30 to 50% of accumulated hepatic triglyceride, indicating that additional mechanisms must be involved in the production of the triglyceride-rich fatty liver in response to tetracycline.     

Different expression pattern of hepcidin genes in the liver and pancreas of C57BL/6N and DBA/2N mice

BACKGROUND/AIMS: Male C57BL/6 and DBA/2 mice differ in their liver iron content. The aim of this study was to examine possible differences in the expression of hepcidin genes (Hamp and Hamp2) between the two strains. METHODS: Hepatic mRNAs were quantified by real-time PCR. RESULTS: Ferroportin1, transferrin receptor 2 and HAMP mRNA levels displayed no significant strain differences. However, HAMP2 mRNA levels were higher in DBA/2N mice. In both strains, HAMP2 mRNA content was sex-dependent, with higher values in female animals. Both hepatic HAMP and HAMP2 mRNA levels were elevated by iron overload, but treatment with lipopolysaccharide increased only HAMP mRNA. Lipopolysaccharide also elevated the amount of HAMP mRNA in the pancreas, while pancreatic HAMP2 mRNA levels were decreased. Sequence analysis of hepcidin amplicons from DBA/2N mice predicted an Asn-->Lys substitution at position 73 of the HAMP peptide and a Ser-->Phe substitution at position 76 of the HAMP2 peptide. CONCLUSIONS: Hepatic Hamp2 expression displays considerable strain- and sex-dependent variation. Lipopolysaccharide increases expression of Hamp both in the liver and pancreas, but Hamp2 does not respond to lipopolysaccharide treatment. The significance of the amino acid substitutions in hepcidin peptides in DBA/2N mice is at present unknown.     

Role of neutrophils in a mouse model of halothane-induced liver injury

Drug-induced liver injury (DILI) is a major safety concern in drug development. Its prediction and prevention have been hindered by limited knowledge of the underlying mechanisms, in part the result of a lack of animal models. We developed a mouse model of halothane-induced liver injury and characterized the mechanisms accounting for tissue damage. Female and male Balb/c, DBA/1, and C57BL/6J mice were injected intraperitoneally with halothane. Serum levels of alanine aminotransferase and histology were evaluated to determine liver injury. Balb/c mice were found to be the most susceptible strain, followed by DBA/1, with no significant hepatotoxicity observed in C57BL/6J mice. Female Balb/c and DBA/1 mice developed more severe liver damage compared with their male counterparts. Bioactivation of halothane occurred similarly in all three strains based on detection of liver proteins adducted by the reactive metabolite. Mechanistic investigations revealed that hepatic message levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta); IL-6, and IL-8 were significantly higher in halothane-treated Balb/c mice compared to DBA/1 and C57BL/6J mice. Moreover, a higher number of neutrophils were recruited into the liver of Balb/c mice upon halothane treatment compared with DBA/1, with no obvious neutrophil infiltration detected in C57BL/6J mice. Neutrophil depletion experiments demonstrated a crucial role for these cells in the development of halothane-induced liver injury. The halothane-initiated hepatotoxicity and innate immune response-mediated escalation of tissue damage are consistent with events that occur in many cases of DILI. In conclusion, our model provides a platform for elucidating strain-based and gender-based susceptibility factors in DILI development.     

Indexing Withdrawal in Mice: Matching Genotypes for Exposure in Studies Using Ethanol Vapor Inhalation

Withdrawal Seizure-Prone (WSP) and -Resistant (WSR) mice have been bidirectionally selected for severity of handling-induced convulsions (HIC) following withdrawal from 72 hr of chronic ethanol vapor inhalation. During selection, daily injections of the alcohol dehydrogenase inhibitor, pyrazole, were used to enhance and stabilize blood ethanol concentrations (BEC). After 26 generations of selection, WSR mice show lower withdrawal BEC than WSP mice exposed to the same ethanol vapor concentrations. Because it is desirable to compare mice maintained at the same BEC to assess correlated responses to selection, this has necessitated exposing WSR mice to higher ethanol vapor concentrations than WSP mice to achieve matched chronic BEC. The experiments reported herein demonstrate two methods for producing matched withdrawal BEC: (1) by exposing mice to the same ethanol vapor concentration and varying the pyrazole dose; and (2) by administering only ethanol at different vapor concentrations and selecting some mice with approximately the same BEC. When exposed to the same ethanol vapor concentration, WSR mice given 1.0 mmol/kg pyrazole had withdrawal BEC equivalent to WSP mice given 0.75 mmol/kg pyrazole. However, WSP mice had much more severe withdrawal HIC than WSR mice. WSP and WSR mice metabolized ethanol at the same rate following withdrawal. The basis for the differential effectiveness of pyrazole is unknown. We also exposed mice to higher ethanol vapor concentrations in the absence of pyrazole. By exposing WSR mice to higher concentrations than WSP, roughly equivalent BEC on withdrawal was achieved. Because BEC are more variable in the absence of pyrazole, it was necessary to select animals of each genotype to achieve relatively matched BEC. Again, WSP mice had much more severe HIC on withdrawal than WSR.     

Revisiting Intragastric Ethanol Intubation as a Dependence Induction Method for Studies of Ethanol Reward and Motivation in Rats

Background: The purpose of this study was to re‐examine intragastric ethanol intubation as a dependence induction method that effectively induces physical dependence upon ethanol over a short time period, is devoid of intrinsic stress artifacts, inexpensive, and easy to implement. Methods: Male Wistar rats were subjected to ethanol dependence induction via intragastric ethanol intubation. Ethanol solution (final concentration 20%, made up in a dietary liquid vehicle consisting of powdered milk, sucrose, and water) was intubated 4 times per day, at 4‐hour intervals, for 6 consecutive days (for a total of 10 g/kg/day). The utility of this procedure was evaluated for inducing physical dependence, determined by daily and final withdrawal ratings. Anxiety‐like behavior associated with ethanol dependence history was examined using the elevated plus‐maze (EPM) test, conducted 5 days after ethanol withdrawal. To evaluate whether potential stress‐like effects of intragastric intubation per se produce lasting effects on behavior, experimentally naive rats were compared with vehicle‐intubated rats for anxiety‐like behavior on the EPM. Results: Blood alcohol levels reached stable levels between 200 and 250 mg%, measured 1 hour after the second and third ethanol intubation on days 2, 4, and 6. Ethanol‐treated rats developed significant somatic withdrawal signs, recorded daily between 10 and 12 hours after the last ethanol administration. At 5 days postwithdrawal, ethanol‐treated rats showed significant anxiety‐like behavior, measured by decreased open arm time and open arm entries on the EPM, compared with vehicle controls. Additionally, ethanol postdependent rats showed decreased open arm time compared with experimentally naive rats. EPM performance did not differ between vehicle‐intubated and naive rats. No withdrawal seizures were observed and mortality rate was near zero. Conclusions: These findings suggest that intragastric ethanol administration produces a behavioral profile consistent with ethanol dependence (i.e., significant withdrawal signs after termination of ethanol exposure and elevated anxiety‐like behavior persisting beyond completion of physical withdrawal), and that the intubation procedure itself does not produce lasting nonspecific anxiety‐like effects. Thus, under the conditions employed here, this procedure provides an effective tool for inducing and evaluating the consequences of ethanol dependence in animal models of ethanol reward and motivation.     

Differential contributions of C3, C5, and decay-accelerating factor to ethanol-induced fatty liver in mice

BACKGROUND AND AIMS: The complement pathway is an important component of the innate and adaptive immune response. Here we tested the hypothesis that activation of complement is required for development of ethanol-induced fatty liver. METHODS: Wild-type mice and mice lacking the third (C3) or fifth (C5) components of the complement activation pathway, as well as mice lacking decay-accelerating factor (CD55/DAF), a complement regulatory protein, were fed Lieber-DeCarli ethanol-containing diets for 6 weeks or pair-fed control diets. RESULTS: Ethanol feeding to wild-type mice increased C3a in plasma. Wild-type and C5-/- mice fed the ethanol diet developed hepatic steatosis characterized by microvesicular and macrovesicular lipid accumulation and increased triglyceride content. C3-/- mice did not develop steatosis, while CD55/DAF-/- mice accumulated even more hepatic triglyceride after ethanol feeding than wild-type mice. Levels of serum alanine aminotransferase and hepatic tumor necrosis factor alpha, indicators of hepatocyte injury and inflammation, respectively, were increased in wild-type and CD55/DAF-/- mice but not in C5-/- mice after ethanol feeding. In contrast to the protective effect of C3-/- against ethanol-induced steatosis, levels of both alanine aminotransferase and tumor necrosis factor alpha were increased in C3-/- mice after ethanol feeding. CONCLUSIONS: Here we have identified several elements of the complement system as important contributors to ethanol-induced fatty liver. C3 contributed primarily to the accumulation of triglyceride in the liver, whereas C5 was involved in inflammation and injury to hepatocytes. Further, the absence of CD55/DAF exacerbated these responses, suggesting that CD55/DAF serves as a barrier to ethanol-induced fatty liver.