Gerald E. McClearn

Enzyme activities and ethanol preference in mice

Alcohol dehydrogenase and aldehyde dehydrogenase, the two principal enzymes of alcohol metabolism, were assayed in the livers of the inbred mouse strains C57BL/6J and DBA/2J. Previous work has shown that animals of various C57BL substrains prefer a 10% ethanol solution to water in a two-bottle preference test, and that animals of various DBA/2 substrains avoid alcohol. In the present study, C57BL/6J mice were found to have 300% more aldehyde dehydrogenase activity than DBA/2J mice and 30% more alcohol dehydrogenase activity. The F1 generation is intermediate to the parents in preference for the 10% alcohol solution and is also found to possess intermediate levels of alcohol and aldehyde dehydrogenase activity. These experiments suggest a systematic relationship between the behavioral trait of ethanol preference and the activity of aldehyde dehydrogenase and a similar but much less pronounced relationship with alcohol dehydrogenase.

Interrelationships of alcohol consumption, actions of alcohol, and biochemical traits

Voluntary alcohol consumption, acute tolerance, and central nervous system (CNS) sensitivity to ethanol are potentially informative measures concerning human alcoholism. Little is understood regarding the associations among these parameters or between these traits and neurochemical processes such as brain protein or brain enzyme activities. A powerful strategy is to assess a large number of characteristics simultaneously on all individuals as a heterogeneous sample. This permits rapid screening of a large number of variables with respect to their interrelationships. Identification can thus be made of those variables that are elements of the caudal nexus, and subsequent experimental research can attack the problem of identifying mechanisms. The present study employed mice from the HS/Ibg stock which is maintained by systematic random mating to assure genetic heterogeneity. The results demonstrate that voluntary ethanol consumption and acquisition of acute tolerance to ethanol were positively associated, whereas these measures were not significantly related to CNS sensitivity to ethanol. In addition, ethanol preference was inversely related to soluble brain protein. The activities of the soluble enzymes from brain, aldehyde reductase and glucose-6-phosphate dehydrogenase, were not significantly associated with ethanol preference, acquisition of acute tolerance, or CNS sensitivity to ethanol. Unexpectedly, more than 30 percent of the variance in voluntary alcohol consumption could have been predicted from the measurements of acquisition of acute tolerance, and vice versa.

ALCOHOL DEHYDROGENASE ACTIVITY AND PREVIOUS ETHANOL CONSUMPTION IN MICE.

PREVIOUS investigations1,2 have shown marked strain differences in ethanol preference of mice, and a correlation between strain means for alcohol preference and for liver alcohol dehydrogenase (ADH) activity has been described3. Further investigation of this association between ADH and preference is needed to clarify the causal relationships, however.

A twin methodology for the study of genetic and environmental control of variation in human smoking behavior.

A method is presented for partitioning the variance associated with human smoking behavior into additive genetic, nonadditive genetic, prenatal environmental, postnatal familial environmental, and postnatal extrafamilial environmental components. Estimations can also be made of additive genetic and residual correlations between spouses and of the correlation between parental additive genetic effect and progeny nonadditive genetic and environmental effect. The variance estimates are free of the biases that might result from these correlations. The statistical genetic analysis is being applied to a large group of MZ and DZ twins, their spouses, and their adult children who live in southern Sweden. Blood samples from each subject will be used to identify their genetic constitution for a number of biochemical polymorphisms, some of which may be considered a priori to have possible relationships to smoking. Associations and genetic linkages between biochemical marker loci and quantitative behavioral traits will be sought. Traits of interest include a wide array of tobacco-use variables, motives for smoking, personality and cognitive variables, and other variables associated with drug use and health. Zygosity determinations based on biochemical polymorphisms have indicated MZ to DZ and DZ to MZ misclassification rates of 0% and 6.15%, respectively, when based solely on external morphology and questionnaire data. The nonpaternity ratio of the fathers with respect to their supposedly biological children is estimated to be 0.28%. Gene frequency estimates for 21 marker loci show that the sample of twins and their relatives is quite representative of the Swedish population at large. All loci were in Hardy-Weinberg-Castle equilibrium, with no evidence of assortative mating for biochemical traits. The MZ twins are significantly more concordant than the DZ twins with respect to whether they have ever had a smoking habit.

GENETICS AS A TOOL IN ALCOHOL RESEARCH

A recurrent theme at this conference has been that nature and nurture cannot profitably be viewed as alternative categories, one and only one of which can be invoked with respect to a particular characteristic. An appreciation of the joint action of genetic factors and environmental ones can but accelerate the understanding of etiological forces in alcoholism. In addition to this very important improvement in theoretical perspective, the study of genetic factors in alcohol preference, sensitivity to alcohol, and other alcohol-related behavior in experimental animals has made available a very powerful tool to those researchers who employ animal models in studying environmentally or experientially derived aspects of alcohol behavior. Briefly stated, genes may be employed as variables, to be controlled or manipulated as other variables. This control derives basically from the ability of the experimenter to assign matings. Two types of mating procedure have been used most extensively with respect to alcohol research, inbreeding and selective breeding, A third procedure, random mating, has been used hardly at all as yet, although its potential utility is a( least equal to that of inbreeding and selective breeding.

Ethanol Consumption: Selective Breeding in Mice

From a foundation stock of HS/Ibg mice, mass selection has yielded highethanol acceptance (HEA) and low-ethanol acceptance (LEA) lines with distributions which show little overlap in the 10th generation. Divergence has been systematic and the estimate of the realized heritability after 10 generations of selective breeding is 0.21±0.04. Low to moderate correlations among three methods of measuring alcohol consumption, including the one on which selection was based, were shown in an F2 generation derived from C57BL/Ibg and C3H/Ibg mice.

Assortative mating in mice. I. Female mating preference.

Females of two inbred strains ofMus musculus domesticus, C57BL/Ibg and DBA/Ibg, were allowed to choose between two males, one of each strain, who were restrained within their cages. Females of both strains preferred to spend more time with, and to mate with, the males of the opposite strain rather than with males of their own strain. No major strain difference for the degree of preference was found. Preference was also unchanged between trials. There is a general lack of correlation between trials which is explained in terms of the nature of the variance in inbred strains. Female mating preference may have an evolutionary significance in reducing inbreeding.

Effects of chronic administration of tobacco smoke to mice: Behavioral and metabolic measures

Tobacco smoke was administered to male and female mice of four inbred strains and two lines selectively bred for high activity (HA) or low activity (LA) in an open field. Administration occurred during 14 daily 10-min pretreatment sessions in a box filled with smoke from a nonfiltered cigarette (2 mg nicotine/ cigarette; average density, 750 ppm carbon monoxide). When open-field activity was subsequently measured in the absence or presence of smoke (average density, 150 ppm carbon monoxide), pretreated mice had significantly lower activity scores than controls. Comparisons of open-field activity scores under smoke-present and smoke-absent conditions revealed that effects of this acute exposure were dependent upon genotype: C3H/2Ibg activity was almost tripled in the presence of smoke; DBA/2Ibg activity was increased; but HA, LA, and C57BL/6Ibg activity scores were depressed. As measured by open-field activity, development of tolerance to the effects of acute exposure to tobacco smoke after chronic pretreatment was also genotype-dependent.Significant genotypic differences were found for nicotine remaining in liver, brain, and blood samples when mice were sacrificed 2.5 or 5 min after a weight-specific injection of radiolabeled nicotine. Tissue nicotine levels were also related to sex, time after injection, and pretreatment interactions with genotype. Strong positive correlations were found between the measures of brain and liver levels at 5 min after injection and the behavioral measure of open-field activity under the smoke-present condition.

The use of strain rank orders in assessing equivalence of techniques

In situations where effects of previous testing make it impossible to compare putativety equivalent forms of a behavioral test, inbred strains may be useful. To a considerable extent, animals from an inbred strain represent replicate individuals. Different naive samples from the same strain may be tested in different forms of a test. There is presumptive evidence that the two forms arc assessing similar functions if samples from different strains that differ on the behavior give consistent ordinal rankings. An illustration is given of the use of this approach in assessing methods of measuring alcohol preference in mice.

Assortative mating in mice. II. Strain differences in female mating preference, male preference, and the question of possible sexual selection

Females ofMus musculus domesticus from the inbred strains DBA/Ibg, C57BL/Ibg, C57BL/J, BALB/Ibg, and C3H/J were tested for mating preference choice between males from their own strain and males from another strain. A general consistency with previously reported results was found, in that the females preferred to associate and mate with males of the opposite strain. However, no preference was found in the case of two strains: BALB/Ibg and C3H/J. Also, C57BL/Ibg females, which generally displayed mating preference for the opposite strain, did not show any preference when allowed to choose between C57BL/Ibg and BALB/Ibg males. The possible effect of male differential attractiveness or sexual ability (sexual selection) on the determination of female mating preference is excluded since no strain of males had an advantage when “neutral” females from a heterogeneous strain were tested for mating preference. The male mating preference of the strains DBA/Ibg and BALB/Ibg was tested in a situation set up to reduce possible bias from the females differential receptiveness. As in the previous experiment, the males did not seem to have any mating preference.