Dissociation between the time course of ethanol and extracellular dopamine concentrations in the nucleus accumbens after a single intraperitoneal injection.

BACKGROUND: Dopamine release in the nucleus accumbens has been linked to the reinforcing effects of ethanol, but the time course or relationship of this response to ethanol concentrations in the brain has not been studied. METHODS: Various doses of ethanol (0-2.0 g/kg) were administered intraperitoneally to male Sprague Dawley rats, and dopamine and ethanol were simultaneously analyzed in dialysate samples from the nucleus accumbens. A separate study to compare the ethanol-induced dopamine response in male and female rats was carried out by using a 1 g/kg intraperitoneal dose of ethanol. RESULTS: In male rats, 1 and 2 g/kg ethanol significantly increased dialysate dopamine by 40% over basal, whereas 0.25 and 0.5 g/kg ethanol produced a nonsignificant 20% increase. Dialysate ethanol concentrations exhibited a curvilinear decline after reaching peak levels for the lower doses but showed a linear decrease after 1 and 2 g/kg. There was a dissociation between the time courses of extracellular dopamine and ethanol after 1 and 2 g/kg ethanol treatment. The dopamine response returned to basal within 90 min, whereas the ethanol concentrations remained elevated. In a separate study that compared male and female rats, the ratio of the dopamine response over basal to the dialysate ethanol concentrations was significantly decreased at 60 min after an injection of 1 g/kg. However, there were no differences between males and females. CONCLUSIONS: The dissociation between dopamine and ethanol levels may reflect the development of acute tolerance to ethanol-induced dopamine release in the nucleus accumbens within the time course of a single acute injection. Given the strong links between dopamine and ethanol reinforcement, our findings may be relevant for understanding the time course of ethanol's reinforcing effects in vivo.

Gender differences in blood levels, but not brain levels, of ethanol in rats.

Female rodents tend to drink more alcohol than males, a difference that emerges at puberty and appears to vary over the female estrous cycle. In addition, male and female rodents display different responses to alcohol; for example, female rats are reported to have faster elimination rates than males. We were interested in whether circulating ovarian hormones influence alcohol distribution to or elimination from the brain of rats, which might explain observed differences in drinking behavior. We administered 0.8 g/kg of ethanol via intraperitoneal injection to age-matched male and female Sprague-Dawley rats. Extracellular brain ethanol was sampled using microdialysis, and vascular ethanol concentrations were determined via tail blood collection, in two separate experiments. Ethanol pharmacokinetic parameters were calculated for both compartments. There were no differences in pharmacokinetic parameters due to gender or estrous cycle stage in brain ethanol concentration profiles. There were, however, differences in blood ethanol profiles: females showed faster elimination rates and a smaller area under the ethanol concentration versus time curve than males. In addition, the maximum concentration varied significantly across the estrous cycle. These results suggest that (1) circulating ovarian hormones do not influence alcohol distribution to the brain, but do influence distribution to more peripheral tissues such as the tail; and (2) apparent differences in tail blood alcohol levels may not reflect differences in brain levels.

Determination of benzidine in the food colours tartrazine and sunset yellow FCF, by reduction and derivatization followed by high-performance liquid chromatography.

Free and bound benzidine, a non-sulphonated aromatic amine (NSAA), were determined in the food colours tartrazine and sunset yellow FCF. Bound amines were released by reducing with sodium dithionite, then total NSAAs were extracted into chloroform, transferred to aqueous acid solution and diazotized with sodium nitrite before coupling with 2-naphthol-3,6-disulphonic acid, disodium salt (R-salt). Coloured benzidine and aniline derivatives (BZDRS and ANRS) were analysed using reversed-phase ion pair high-performance liquid chromatography (HPLC) and an absorbance detector set at 548 nm. Levels of total benzidine were similar to those reported in studies conducted in the USA, and ranged from < 5 to 270 ng/g. Total aniline was also determined (0.2-188 micrograms/g). Recoveries of benzidine with this method were found to be lower than expected (average ca 46%), but were reproducible. Detection limits were 15-20 ng BZDRS/g (3-4 ng benzidine/g). Mass spectrometry (LC-MS) was evaluated for identifying and determining purity of the standards, but difficulties were encountered when the methodology was applied to commercial samples.

Sex differences in voluntary drinking by Long Evans rats following early stress.

Models for early stress and voluntary drinking were used to determine the contribution of early stress to increased intake of alcoholic beverages during puberty and adulthood. Newborn litters of Long Evans rats were: (1) stressed by daily separation from the mother for 15 min/day on days 1 to 7 of life ["handled" (H)]; or (2) left untouched with the mother on days 1 to 7 of life ["nonhandled" (NH)]. All animals were weaned on day 22, separated by sex (M and F), and caged individually with an assignment of 10 animals per sex per treatment group (H and NH). From 25 to 85 days of age, all animals were given free access to beer containing 5% ethanol (v/v), water, and regular laboratory food. Beer, food, and water intake was measured daily at the same time each day, and animals were weighed weekly. HM had greater ethanol intake and preference for ethanol during the peripubertal period (days 32 to 45), compared with all other groups. There were no differences in ethanol intake between NHF and NHM. HM had greater ethanol preference than HF on 22 of the 60 drinking days. HF consumed the same amount of water as the males and significantly greater amounts of water than NHF on 28 of the 60 drinking days. HM had greater ethanol preference than NHM on 8 of the 60 drinking days. From day 75 to day 85, HF had greater ethanol intake than HM, and NHF had greater ethanol intake than NHM. There were no differences in body weights of HF and NHF throughout the study. Growth of HM lagged behind NHM into adulthood. Early stress of males was linked to increased ethanol intake during the peripubertal and adult periods and stunted body growth into adulthood. Early stress of females was linked to polydipsia (water) throughout development and continuing into adulthood, and to increased alcohol intake in adulthood.

Physical and motor development in male and female rat offspring prenatally exposed to gamma radiation.

The effects of radiation on physical and motor development of male and female rats exposed to ionizing radiation in utero were studied. Rats were exposed to three different doses of radiation (150 rad, 15 rad and 6.8 rad, considered as high, moderate and low doses of radiation respectively) on the 20th day of prenatal life. Exposure to 150 rad contributed to significantly lower body weights of both male and female rat offspring. Upper jaw tooth eruption was delayed in 150 rad treated male offspring, as well as in 15 rad and 150 rad treated female offspring. Cliff-avoidance response was delayed in 6.8 rad, 15 rad, and 150 rad treated male offspring; and 150 rad treated female offspring. Lower jaw tooth eruption, eye opening, and crawling were not affected by radiation in male or female animals. Results indicate that radiation affected the developmental parameters of both male and female rat offspring, and sex of the offspring played no role in the magnitude of radiation induced damages.

Sex differences in alcohol preference and drinking patterns emerge during the early postpubertal period.

Young male and female Sprague-Dawley rats (30 days old) were assigned randomly to three treatment groups: (1) alcohol treatment--received beer with 5% ethanol added, food, and water ad libitum; (2) pair-fed treatment--received nonalcoholic beer plus sucrose and food to match intake by the alcohol-treated animals; and (3) control treatment--received food and water ad libitum. Animals were tested for alcohol preference for 24 hr and then received their assigned treatments for a period of 30 days, followed by a period of abstinence before alcohol preference testing again at 74 days of age. Males given free access to beer and water did not drink large quantities of beer. Females given free access to beer and water drank a lot of beer on the first day, but decreased intake until approximately 52 days of age. A developmental change in young female rats at approximately 52 days of age resulted in increased voluntary ethanol intake, possibly caused by hormonal changes associated with the establishment of estrous cycles. When the animals were tested for alcohol preference at 74 days of age after a period of abstinence, males and females in the pair-fed group had greater alcohol preference than animals in the other groups. Females in the pair-fed group had greater alcohol intake based on body weight than males in the pair-fed group and males and females in all other groups. These results provide insight into sex differences in the development of voluntary drinking behavior and responses of drinking behavior to the early stress of pair-feeding.

High-performance liquid chromatographic separation of carminic acid, alpha- and beta-bixin, and alpha- and beta-norbixin, and the determination of carminic acid in foods.

During a study of natural food colours, a simple and reliable high-performance liquid chromatography system was developed for use with cochineal and annato. An isocratic mobile phase, consisting of methanol and 6% aqueous acetic acid, resolved bixin and norbixin, while a gradient system was used to separate carminic acid and the annato compounds. The carminic acid contents of cochineal extract, carmine and carmine hydrosoluble were determined using an isocratic mobile phase (40:60, v/v). The detection limit for carminic acid in the various products was approximately 100 ng/g. Carminic acid was determined quantitatively in fruit beverages, yogurt and candies. It was demonstrated that, because of decomposition, carminic acid was not suitable for use in candies when manufacturing temperatures above 100 degrees C were required. Most membrane filters are not suitable for use with cochineal solutions, but a cellulose membrane filter did not adsorb carminic acid and was used successfully to remove impurities from water-based cochineal products and food extracts containing carminic acid.

Alcohol and the brain: what's NO got to do with it?

A potential role for nitric oxide in alcohol-induced changes in brain function is discussed. Chronic alcohol exposure may lead to excitotoxicity partially due to increased levels of nitric oxide (NO). Excessive NO has been linked to cytotoxicity in neurons, glia and myelin. Cytokines produced in response to cell injury may trigger increased production of NO. These events may be involved in alcohol-induced brain damage. Formation of NO has recently been linked to increased preference for and tolerance to alcohol. A hypothesis for prevention and treatment of alcohol-induced brain damage, and craving and alcohol intake by alcoholics is proposed.

Gender differences in animal studies. Implications for the study of human alcoholism.

The reasons for gender differences in alcohol intake, responses to alcohol, and consequences of alcohol abuse in humans and in animals are poorly understood. Animal models for the study of alcoholism have been focused primarily on the study of male rodents, although researchers have observed that female rodents drink more alcohol than males and have sex-related differences in drinking patterns and responses to alcohol. In humans, the difference between the sexes is the opposite of rodents, with males drinking more than females. These results suggest differences between the sexes and differences between humans and rodents in drinking behavior and responses to alcohol which may be based on a complex interaction of social, genetic, hormonal, neurobiologic, and environmental factors. Four new studies are introduced to identify sex-distinct genetic influences in alcohol-related phenotypes, sex-based differences in behavioral responses to alcohol, sex differences in responses of brain reward systems to alcohol, and interactions of the anxiolytic effects of alcohol with steroids and the estrous cycle.

Determination of annatto in high-fat dairy products, margarine and hard candy by solvent extraction followed by high-performance liquid chromatography.

Utilizing solvents such as ethanolic aqueous ammonia, petroleum ether, hexane and chloroform, annatto components alpha- and beta-norbixin and alpha- and beta-bixin were extracted from cheese, butter, margarine and hard candy. After transferring the extract into a solution of aqueous acetic acid in methanol, bixin and norbixin were determined quantitatively using high-performance liquid chromatography (HPLC) and an absorbance detector set at 500 nm. Recovery of norbixin from spiked cheese samples averaged 92.6% over a range of 1 to 110 micrograms/g. Commercial cheese samples were found to contain 1.1-68.8 microgram/g total norbixin, and two samples also contained 5.1-5.6 micrograms/g total bixin. Samples of uncoloured butter were spiked with bixin and recovery averaged 93.2% over a range of 0.1 to 445 micrograms/g. Levels of 0.2 microgram/g total bixin and 0.91 microgram/g total norbixin were found in one commercial butter sample; the others contained trace levels of both compounds. Hard candies were prepared in the laboratory and recovery studies conducted. Recovery of norbixin averaged 88%.

Alcohol and white matter development--a review.

This is a review of the literature on the effects of alcohol on white matter development. For many years, human and animal studies have reported the vulnerability of developing white matter to the effects of alcohol. However, until recently, studies on alcohol and white matter were limited by existing technology. New technology documenting the presence of neurotransmitter receptors and ion channels on glial cells now provides a new focus for research on alcohol and white matter development. New research using new technology should enlarge our knowledge of the role of glial cells in brain damage associated with alcohol exposure during development.

Gender differences in the brain: implications for the study of human alcoholism.

Gender differences in alcohol intake and response to alcohol may be influenced by basic variations in the organization and modulation of male and female brains. Although a number of genetic, social, environmental, and metabolic factors have been proposed to explain the gender differences observed in risk for alcoholism, alcohol intake, and medical consequences of excessive alcohol intake, very little attention has been given to the role of gender differences in the brain regarding alcohol use. Recent evidence documents the influence of neurosteroids on neurotransmitter activity in the brain and the impact of alcohol on neurosteroid levels. Neurosteroids are found in different levels in males and females during development and throughout life, depending on factors such as age, stage of development, estrous and menstrual cycles, and stress. This study discusses the hypothesis that many of the gender differences observed concerning alcohol use and misuse are determined by gender differences in the brain, which in turn differentially influence the behavioral and neurochemical responses of males and females to alcohol.

Locomotion and physical development in rats treated with ionizing radiation in utero.

Effects of ionizing radiation on the emergence of locomotor skill, and physical development were studied in laboratory rats (Fisher F-344 inbred strain). Rats were treated with 3 different doses of radiation (150 rad, 15 rad, and 6.8 rad) delivered on the 20th day of prenatal life. Results indicated that relatively moderate (15 rad) to high (150 rad) doses of radiation had effects on certain locomotion and physical development parameters. Exposure to 150 rad delayed pivoting, cliff-avoidance, upper jaw tooth eruption, and decreased body weights. Other parameters, such as negative geotaxis, eye opening, and lower jaw tooth eruption were marginally delayed in the 150 rad treated animals. Exposure to 15 rad delayed pivoting and cliff-avoidance.

Brain myelination in rats treated with ionizing radiation in utero.

Effects of ionizing radiation on brain myelination and some physical development parameters were studied in laboratory rats (Fisher F-344 inbred strain). Rats were treated with three different doses of radiation (150 rad, 15 rad, and 6.8 rad) delivered on the 20th day of prenatal life. Exposure to 150 rad reduced body, brain, ovary, kidney, heart and spleen weights. Prenatal exposure to 150 rad of radiation reduced the cerebral cortex weight by 22 percent at 30 days of age, and 20 percent at 52 days of age which caused a reduction in cerebral cortex myelin content by 20 and 23 percent at the ages of 30 and 52 days respectively. This dose did not affect the myelin content of the cerebellum or the brain stem, or the myelin concentration (mg myelin/g brain tissue) of the cerebral cortex, cerebellum, and the brain stem. The cerebral cortex weight of the 15 rad treated rats was reduced at the age of 30 days. Exposure to 15 rad, and 6.8 rad did not affect either the myelin content or the myelin concentration of these brain areas.

Sex differences in pattern of drinking.

Drinking patterns of male and female Long-Evans rats were compared during a 15-day drinking period. All animals were tested for preference for alcohol for 24 h during which food, water, and beer containing 5% ethanol were freely available. Animals drinking 50 ml or more of beer were chosen for the experiments. On days 1-5, animals were offered food, water, and beer containing 5% ethanol (v/v). On days 6-15, the concentration of ethanol in the beer was doubled to 10% (v/v). Preference ratios (beer/total fluid) were higher for females than males, and females consumed more grams of alcohol per unit of body weight. When alcohol concentration was doubled, females increased alcohol intake (g/kg), while males tended to titrate alcohol intake to levels consumed at 5% concentration. Female patterns of drinking differed from male patterns of drinking.

Alcohol, nitric oxide, and neurotoxicity: is there a connection?--a review.

A hypothesis is presented to explain the influence of alcohol on glutamate generated excitotoxicity. Chronic alcohol exposure is reported to increase glutamate-N-methyl-D-aspartate (NMDA) receptors and calcium ion channel activity, resulting in the neurotoxicity and seizure activity associated with alcohol withdrawal in certain persons. Recent information indicates that nitric oxide is responsible for the neurotoxicity associated with excessive glutamate stimulation of NMDA receptors. Thus, it is hypothesized that nitric oxide is involved in producing the neurotoxicity and cell disturbances associated with chronic alcohol exposure.

Determination of total non-sulphonated aromatic amines in soft drinks and hard candies by reduction and derivatization followed by high-performance liquid chromatography.

Utilizing elements of methodology developed previously for food colours, total free and bound non-sulphonated aromatic amines (NSAA) were determined in commercial samples of soft drink beverages and hard candies. Bound amines in the samples were reduced using sodium dithionite, then total NSAA were extracted into chlorofom, transferred to aqueous acid solution and diazotized with sodium nitrite before coupling with 2-naphthol-3,6-disulphonic acid, disodium salt (R-salt). The coloured derivatives were analysed using reversed-phase ion pair high-performance liquid chromatography (HPLC) and an absorbance detector set at 512 nm. Solid phase extraction cartridges were utilized for extraction and clean-up of the food colours present in the sample, and the concentration of each dye was determined quantitatively using HPLC and absorbance detector wavelengths of 426, 516 or 625 nm. Levels of total NSAA were compatible with those observed previously in food colours. Commercial soft drinks were found to contain (expressed in terms of total free plus bound NSAA in the beverage) 0.19-12.6 ng/ml of aniline, 0.83-8.25 ng/ml 1-naphthylamine and 0.62-1.12 ng/ml 2-naphthylamine. Levels of 0.66-9.15 ng/g of aniline and 2.48-10.6 ng/g 1-naphthylamine were found in commercial samples of hard candies. Bound NSAA in hard candies appeared to survive the manufacturing process. Recoveries averaged 96.9% for tartrazine and 89.6-97.2% for the bound amines when hard candies were prepared in the laboratory.

Alcoholic and nonalcoholic beer drinking during gestation: offspring growth and glucose metabolism.

Female Long-Evans rats were allowed voluntary access to beer, food and water for 52 days prior to mating, throughout mating and throughout gestation, and were compared to animals pair-fed nonalcoholic beer and to regular controls. Alcoholic beer drinkers gained more weight during gestation than drinkers of nonalcoholic beer. Significant hypoglycemia was observed in the newborn male offspring of alcoholic beer drinkers. At 20 days of age, all animals responded normally to glucose tolerance tests. At 20 days of age, liver weights of offspring of beer drinkers (alcoholic and nonalcoholic) were enlarged; pancreas weights of alcoholic beer drinkers were increased. At 65 days of age, body weights of male offspring of alcoholic beer drinkers were low. These results indicate sex differences in response to maternal beer drinking, and suggest some of the observed alterations in development were due to components in beer other than alcohol.

Determination of total non-sulphonated aromatic amines in tartrazine, sunset yellow FCF and allura red by reduction and derivatization followed by high-performance liquid chromatography.

Free and bound non-sulphonated aromatic amines (NSAA) are determined in the food colours tartrazine, sunset yellow FCF and allura red. After reduction of the bound amines with sodium dithionite, the NSAA are extracted into chloroform, then transferred to aqueous acid solution, diazotized with sodium nitrite and coupled with 2-naphthol-3,6-disulphonic acid, disodium salt (R-salt). Reversed-phase ion-pair liquid chromatography and an absorbance detector at 512 nm are used to analyse the coloured derivatives. Samples of dyes were spiked with known amounts of aniline, 1-naphthylamine, 2- and 4-aminobiphenyl, 4-aminoazobenzene, benzidine, p-cresidine or 4-nitro-p-cresidine bound to R-salt. Recoveries averaged 90% in tartrazine, 65% in sunset yellow FCF and 71% in allura red. Detection limits ranged between 2 and 32 ng/g. A survey of 24 commercial samples revealed levels up to 520 micrograms/g total NSAA. The majority of NSAA are bound to the coupling compound during the manufacturing process and less than 7% remain as free amines in the dye.

Voluntary beer drinking by pregnant rats: offspring growth, development and behavior.

A new model for voluntary beer drinking during gestation is presented. Female Long-Evans rats voluntarily drank 56 ml beer per day (pregestation) and 66 ml beer per day (gestation), resulting in an ethanol intake of 9.5 g/kg/day (pregestation) and 9.0 g/kg/day (gestation). Peak blood alcohol levels of dams were 192.5 mg/dl (pregestation) and 157 mg/dl (gestation). Body weights of male and female offspring of beer drinkers were greater than controls. Urinary pH levels were abnormally low at birth, and thymus/body weight ratios were high at birth. Abnormal spleen/body ratio and heart/body ratios were observed in 15- and 29-day-old female offspring of beer drinkers. No hyperactivity or developmental delays were observed. Male offspring of dams in the beer group were hypoactive on a few days of testing. Male and female offspring of beer drinking dams performed better than controls on some tests. This method for voluntary beer drinking by pregnant rats may be useful for identifying the subtle consequences of maternal beer drinking.