Toni Ness Smolen

Blood and brain ethanol concentrations during absorption and distribution in long-sleep and short-sleep mice

It is often assumed that blood ethanol content accurately reflects brain ethanol content. In previous studies we have found that at the time of regaining the righting response blood and brain ethanol levels were identical, and blood ethanol could be used to predict brain ethanol level. It is likely, however that shortly after the administration of ethanol, blood and brain ethanol levels would differ. For this study, venous blood (orbital sinus) and brain ethanol levels were measured in long-sleep and short-sleep mice within the first 30 min following ethanol administration (2.5-6.0 g/kg). Ethanol was administered intraperitoneally or intragastrically. For both lines of mice and for every dose, brain ethanol concentrations were significantly greater (as much as 100 mg/dl) than blood ethanol levels for the first 6 min, and peak blood and brain ethanol levels were reached 4 to 6 min after dosing. Approximately 6 to 10 minutes (depending on dose and line of mouse) was required for blood and brain concentrations to reach equilibrium. At the time of loss of the righting response, brain ethanol levels were significantly higher than blood ethanol levels. These results indicate that within the first 6 min after administration of ethanol, blood ethanol level is not suitable for the assessment of brain ethanol content.

Genetically determined differences in acute responses to diisopropylfluorophosphate

The acute effects of diisopropylfluorophosphate (DFP) were assessed in DBA/2Ibg, C57BL/6Ibg and C3H/2Ibg mice. The DFP was administered by intraperitoneal injection in saline. Brain acetylcholinesterase (AChE) activity was maximally inhibited within 5 min after injection. All mice showed signs of organophosphate intoxication including salivation, lacrimation, diarrhea, respiratory distress, tremor and, at high doses, seizures. The C57BL mice were most susceptible to these effects of DFP. The LD50 values for DFP were 8.0, 7.6, and 6.8 mg/kg for male DBA, C3H, and C57BL mice, respectively. The LD50 values for females were nearly the same. Body temperature and brain AChE activity decreased in a dose-dependent manner following injections of DFP of 3.17, 4.22, 5.28, and 6.33 mg/kg. Maximum temperature depression occurred 2 hours after DFP administration; by 24 hours temperatures had returned to normal except for C57BL mice treated with the highest dose of DFP. The C57BL strain was most susceptible to the DFP-induced hypothermia, the C3H strain was the most resistant, and the DBA strain was intermediate. Maximum temperature depression and residual AChE activity, as measured 24 hours after injection, were linearly related. These strain differences do not seem to be explained easily by a differential inhibition of AChE activity.

Subcellular distribution of hepatic aldehyde dehydrogenase activity in four inbred mouse strains

1. 1. The subcellular distribution of hepatic NAD+-linked aldehyde dehydrogenase (AIDH) was studied in four inbred mouse (Mus musculus) strains: C57BL/6J, DBA/2J, BALB/cJ, and C3H/2Ibg. 2. 2. The distribution pattern of A1DH activity was 70% cytosolic, 20% mitochondrial, and 10% microsomal. Each of the inbred strains showed this same general pattern but significant differences (P < 0.01) were found in the levels of the enzymes among the strains, whether these were expressed as specific (per mg protein) or total (per g liver) activity. 3. 3. Mitochondrial and microsomal enzymes from each of the inbreds were able to catalyze the oxidation of propionaldehyde with NADP+ substituted for NAD+ at 10–20% of the rate with NAD+. In contrast, the only cytosolic enzymes that had activity with NADP+ were those from the DBA/2J strain. 4. 4. It was concluded that the distribution of AlDH in mouse liver is different from that in rat liver and that the differences in enzyme activity among the mouse strains may play some role in their behavioral responses to ethanol.

Sensitivity of inbred and selectively bred mice to ethanol.

The Long-Sleep (LS) and Short-Sleep (SS) mice were bred for differences in sensitivity to ethanol as measured by duration of loss of the righting response (sleep time). The foundation population was a heterogeneous stock (HS) which was derived from a cross of eight inbred strains. Ethanol-induced sleep time and waking blood and brain ethanol levels were measured in the eight inbred strains, LS, SS and HS mice. The C3H and ISBI strains were quite resistant to ethanol as measured by sleep time, and only one, RIII, was very sensitive. Waking ethanol concentrations were similar for all of the inbreds, implying a narrow range of central nervous system sensitivity to ethanol. The HS mice had relatively short sleep times and blood ethanol levels equal to most of the inbred. The LS mice were significantly more, and the SS mice significantly less sensitive to ethanol than any of the inbreds or HS mice. These studies suggest that the extremes of CNS sensitivities to ethanol manifested by the LS and SS mice cannot be traced to any of the inbred strains, and must have arisen through the selection process by changes in allelic frequencies of those genes conferring ethanol sensitivity and resistance.

A strain comparison of physiological and locomotor responses of mice to diisopropylfluorosphosphate

The effects of acute treatment with the organophosphate, diisopropylfluorophosphae (DFP), were studied in three inbred mouse strains. C57BL, DBA and C3H. A battery of physiological and locomotor tests including respiratory rate, heart rate, body temperature, Y-maze activity and rotarod performance was used. Dose-response and time course studies were carried out. Approximately 15 min after injection the animals were markedly affected by the drug with maximal effects occurring approximately 2 hours after injection. Strain comparisons were made at the 2 hr time point. In all strains, males and females were affected about equally except for respiratory rate and rotarod performance in which females were slightly more affected. Strain comparisons revealed that for most of the tests the C57BL mice were most affected by the DFP and the C3H mice were least affected. For the heart rate test the DBA mice were the most sensitive. Previous studies from our laboratory have demonstrated a similar rank ordering of the strains in their responses to oxotremorine and nicotine. The strain differences in response to these agents is not easily explained by differences in number or affinity of brain muscarinic or nicotinic receptors. The genetic influence on cholinergic drug response may involve receptor coupling mechanisms.

Genetic variation in paraoxonase activity and sensitivity to diisopropylphosphofluoridate in inbred mice

The mechanism underlying genetic variation in the acute and chronic responses of mice to diisopropylphosphofluoridate (DFP) are unknown. We investigated whether variation in metabolism of organophosphates by A-esterase, as exemplified by the enzyme paraoxonase, was correlated to the degree of sensitivity to DFP in four inbred mouse strains. LD50s and plasma paraoxonase were measured in each strain. We observed genetic variation in both of these measures, but there was no significant correlation between the two measures. We conclude that plasma paraoxonase activity does not underlie genetic variation in sensitivity to the lethal effects of DFP in mice since it does not determine the degree of sensitivity or resistance to DFP.

Alterations in regional brain GABA concentration and turnover during pregnancy

During pregnancy, mice are more susceptible to flurothyl-induced seizures than are nonpregnant control mice. The potential role of brain GABA in mediating this behavior was examined in the present study. GABA concentrations in the cerebellum, hippocampus, striatum, midbrain, and cortex from individual control, pregnant (days 17-18) and delivery-day Heterogeneous Stock mice were assayed using a fluorometric method. Turnover of GABA was assessed by inhibiting metabolism with aminooxyacetic acid and measuring GABA accumulation over the next 2 h. Steady-state GABA concentrations decreased significantly from control in all brain regions during pregnancy. Reductions in GABA concentrations were approximately 25-30% in the affected regions. At parturition, GABA concentrations in the cerebellum and cortex returned to control levels, but hippocampal, striatal, and midbrain GABA levels remained significantly depressed. All the indices of GABA turnover--first-order rate constant, half-life, initial rate of synthesis, and turnover rate (product of first-order rate constant and initial concentration)--showed a significant reduction in pregnancy, which was continued through the time of delivery in all brain regions except the hippocampus. Half-life values for GABA increased nearly fourfold in the cerebellum and cortex. These results show that there is a significant alteration in GABAergic systems during pregnancy and parturition. We suggest that the reduction in GABA turnover is a compensatory anticonvulsant mechanism to offset the inherent seizure susceptibility brought about by the reduced level of the major inhibitory neurotransmitter in the brain.

Dissociation of decreased numbers of muscarinic receptors from tolerance to DFP

Several studies have demonstrated that chronic treatment with organophosphates, such as DFP, elicits a decreased number of brain muscarinic receptors (measured by the binding of QNB) which has been presented as an explanation for tolerance to the organophosphates. The purpose of the studies presented here was to assess whether graded changes in QNB binding could be attained following different methods of chronic DFP treatment, and whether tolerance to DFP paralleled these changes. Male DBA mice were injected with DFP every 4 days or 2 days for 30 days or daily for 14 days. The animals were subsequently challenged with DFP or the muscarinic agonist, oxotremorine, and respiratory rate, heart rate, body temperature, Y-maze activity and rearing were recorded. Chronic DFP-treated animals were supersensitive to the effects of DFP on respiratory rate, heart rate, and body temperature whereas a modest tolerance to the effects of oxotremorine on respiratory rate, heart rate, and body temperature was seen. Neither tolerance nor supersensitivity were observed for the effects of DFP and oxotremorine on the Y-maze measures. Chronic DFP treatment elicited reduced binding of QNB in striatum, cortex, and hippocampus with the group that had been treated every other day exhibiting the greatest changes. The changes in drug response did not parallel changes in QNB binding which raises questions as to the cause of the reduction in binding.

Developmental expression of cocaine hepatotoxicity in the mouse

Cocaine may be metabolized either by ester hydrolysis to inactive products or by oxidation via a cytochrome P-450 and FAD-monooxygenase pathway to a hepatotoxic metabolite, presumably norcocaine nitroxide. Mice are the species most susceptible to cocaine-induced hepatotoxicity (CIH), and marked strain differences in response have been found. Female mice are very resistant to CIH, whereas males are susceptible, indicating that hormonal factors may be involved. We treated mice of 5 inbred strains with cocaine at three ages: 20 days (weanling), 30 days (adolescent) and 60 days (adult). The CIH response was assessed by measurement of plasma alanine aminotransferase (ALT) activity 18 hours later. For each of the strains females of all three age groups were resistant to CIH, and males did not begin to develop CIH until approximately 30 days of age. The degree of CIH in 30-day-old males was intermediate between the levels found in 20-day-old males and adult males. These data suggest that the enzyme, or enzymes, responsible for the production of the toxic metabolite are absent, or at very low levels, in female and immature male mice, and that they are either inducible by androgens or are repressed by estrogens or progestins. It is possible that these enzymes may be involved in the production of toxic metabolites of compounds other than cocaine.

Developmental profile of hepatic alcohol and aldehyde dehydrogenase activities in long-sleep and short-sleep mice

Ethanol is metabolized primarily in the liver by a cytosolic alcohol dehydrogenase (ADH). The product, acetaldehyde, is metabolized to acetate by nonspecific aldehyde dehydrogenases (AHD). Mouse liver contains five major constitutive AHD isoenzymes: mitochondrial high Km (AHD-1), mitochondrial low Km (AHD-5), cytosolic high Km (AHD-7), cytosolic low Km (AHD-2) and microsomal high Km (AHD-3). The Long-Sleep (LS) and Short-Sleep (SS) mice differ in their sleep time response to ethanol as early as 10 days of age, and this difference increases with increasing age. Age- and genotype-related differences in metabolism could account for the pattern of responses seen in these mice. We measured the activity of hepatic ADH and the five AHD isoenzymes in LS and SS mice from 3 days of age to adulthood to determine if there were differences in the developmental profiles of these enzyme activities. We found no sex differences in the developmental profile of either ADH or AHD, and the LS and SS mice have nearly identical ADH and AHD activities with the possible exception of the high Km mitochondrial enzyme activity between days 3 and 6, and the low Km mitochondrial enzyme between days 28 and 32. Thus, it appears that differences in ethanol or acetaldehyde metabolism do not contribute significantly to the differential sensitivity to ethanol between young LS and SS mice or to the differential sensitivity between young and adult mice.