No effect of albinism on sedative-hypnotic sensitivity to ethanol and anesthetics.

BACKGROUND: Studies using the long-sleep (LS) X short-sleep (SS) (LSXSS) recombinant inbred mice and inbred long-sleep (ILS) by inbred short-sleep (ISS) intercrosses have found genetic linkage between Tyr albinism (c/c) and differential sensitivity to sedative-hypnotic doses of ethanol and general anesthetics. This linkage could be due to a gene or genes near Tyr or Tyr itself. With regard to the latter possibility, the absence of tyrosinase activity (encoded by Tyr) in albinos could alter tyrosine availability and thus the rate-limiting step in catecholamine synthesis. In addition, albinism is associated with altered brain development that could have pleiotropic effects on behavior. Therefore, in this study, we asked whether albinism affects sedative-hypnotic sensitivity. METHODS: Loss of righting reflex (LORR) duration was measured using doses of ethanol (4.1 g/kg), pentobarbital (70 mg/kg), isoflurane (2 g/kg), and etomidate (20 mg/kg) that were previously associated with differential sensitivity of albino versus nonalbino mice. Tyr transgenics (c/c, Tg(Tyr+)) were backcrossed to ISS (c/c) to compare pigmented (c/c, Tg(Tyr+)) and albino (c/c) mice in the context of an ISS-like background. ISS was also crossed with C57BL/6 (B6) mice heterozygous for a spontaneous albino mutation (c2j) to compare pigmented (c/+) and albino (c/c2j) mice. Pigmented B6 (c2j/+ and +/+) and albino B6 (c2j/c2j) mice were also compared (pentobarbital). RESULTS: For each sedative hypnotic, albinism had no effect on LORR duration. Each expected difference was ruled out at the 95% or 99% confidence level. For each sedative hypnotic, males were more sensitive than females even though the effect size was usually smaller than the expected albino effect size, arguing empirically that the inability to detect an albino effect was not due to systematic error or an insufficient number of mice. CONCLUSION: We conclude that the differential sensitivity associated with albinism is most likely due to a gene or genes near Tyr rather than Tyr itself.

Gas chromatographic--mass spectrometric determination of etomidate in mouse brain.

A simple, rapid and reliable method was developed to determine the concentration of etomidate [ethyl-1-(1-phenylethyl)-1 H-imidazole-5-carboxylate] in mouse brain tissue by gas chromatography-mass spectrometry (GC-MS). Ethyl 5-amino-1-phenyl-4-pyrazolecarboxylate was used as internal standard (IS) and liquid-liquid extraction using ethyl ether as the solvent. The method demonstrated excellent recovery (93%) and a linear calibration range of 50-2500 ng/0.2 g. Intra-day accuracy and precision had an error and coefficient of variation of less than 8.7% and 4.2%, respectively. The limit of detection was 1 ng in mouse brain. Our results suggest that this new method is suitable for the quantitative analysis of etomidate in mouse brain tissue.

Gas chromatography-mass spectrometry assay for determination of ketamine in brain.

A sensitive and precise gas chromatography-mass spectrometry method with selected ion monitoring has been developed for determination of ketamine in the brain using chlorpheniramine as an internal standard. The assay is based on the acid extraction of brain homogenate with hexane and ethyl ether with subsequent alkaline ethyl ether extraction. The analytical procedure has a coefficient of variation of 3.0-5.3% and from 3.8 to 6.1% for extraction from water or spiked brain samples, respectively. The lowest detectable level of ketamine was 1 ng in any brain region. This level of detection was used to measure the ketamine concentrations in cerebellum, brain stem, midbrain, hypothalamus, and cortex of C57B1/6 mice at awakening following intraperitoneal injection of a hypnotic dose. The ketamine concentrations in mouse brain were in the range from 41.6 to 48.6 ng/mg of tissue.

Differences in NMDA receptor antagonist-induced locomotor activity and [3H]MK-801 binding sites in short-sleep and long-sleep mice.

Short-Sleep (SS) and Long-Sleep (LS) mice differ in initial sensitivity to ethanol. Ethanol acts as an antagonist at N-methyl D-aspartate receptors (NMDARs). Therefore, we tested whether SS and LS mice also differ in initial sensitivity to NMDAR antagonists. Systemic injection (intraperitoneal) of either the noncompetitive NMDAR antagonist MK-801 (dizocilpine) or the competitive NMDAR antagonist 2-carboxypiperazin-4-yl-propyl-1-phosphonic acid (CPP) produced similar results. At lower drug doses, SS mice showed greater locomotor activation than LS mice; and at higher doses, SS mice continued to be activated whereas LS mice became sedated. Brain levels of [3H]MK-801 were 40% higher in SS, compared with LS, mice. However, blood levels of [3H]MK-801 and [3H]CPP and brain levels of [3H]CPP were similar in the two lines. NMDARs were measured using quantitative autoradiographic analysis of in vitro [3H]MK-801 binding to SS and LS mouse brains. Significantly higher (20 to 30%) receptor densities were observed in the hippocampus and cerebral cortex of SS mice. Our results support the hypothesis that SS and LS mice differ in initial sensitivity to NMDAR antagonists and suggest that the line differences in the dose-response relationships for MK-801- and CPP-induced locomotor activity are qualitatively similar to those reported for ethanol. Differences in pharmacokinetics and number of NMDARs may contribute to, but are unlikely to entirely account for, the differential behavioral responsiveness of SS and LS mice to MK-801 and CPP.

Identification of a genetic region in mice that specifies sensitivity to propofol.

BACKGROUND: Long-sleep (LS) and short-sleep (SS) mice, initially selected for differential sensitivity to ethanol, also exhibit differential sensitivity to propofol. By interbreeding LS and SS mice to obtain progeny whose chromosomes are a patchwork of the LS and SS chromosomes, the authors determined whether differential propofol sensitivity cosegregates with any particular chromosomal region(s). Such cosegregation is the essence of genetic linkage mapping and a first step toward isolating a gene that can modulate propofol sensitivity in mammals. A gene underlying a quantitative trait such as anesthetic sensitivity is commonly called a quantitative trait locus (QTL). METHODS: The propofol dose was 20 mg/kg injected retroorbitally. Sensitivity was measured as the duration of the loss of righting reflex (LORR). The LORR and propofol brain levels at awakening were determined for 24 LSXSS recombinant-inbred (RI) strains, derived by intercrossing LS and SS for two generations followed by >20 generations of inbreeding. A genetic linkage between LORR and an albino mutation on chromosome 7 was investigated further using 164 second-generation progeny (F2s) from intercrossing inbred LS and inbred SS mice, similar to the LSXSS RIs except F2s are not inbred. The linkage between propofol sensitivity and the albino locus also was investigated using additional genetic markers on chromosome 7. Statistical significance was assessed by interval mapping using a regression method for RIs and Mapmaker/QTL (Whitehead Institute, Cambridge, MA) for F2s. RESULTS: Genetic mapping in the LSXSS RIs revealed a QTL tightly linked to the Tyr (albino) locus that accounts for nearly all of the genetic difference in propofol sensitivity between LS and SS mice. Analysis of propofol brain levels at awakening indicated that this QTL results from differential neurosensitivity. Mapping in F2s confirmed the genetic linkage to Tyr. Mice (ISS c/c x C57BL/6 c2j/C) that differed only by an albino mutation at Tyr were not differentially sensitive to propofol. CONCLUSIONS: A single QTL, called Lorp1, underlies most of the genetic difference in propofol neurosensitivity between LS and SS mice. Although this QTL is tightly linked to Tyr, propofol sensitivity is not modulated by albinism. For mapping this QTL, the LSXSS RIs proved to be an especially powerful resource, localizing the candidate-gene region to a 99% confidence interval of only 2.5 centimorgans.

Strain distribution patterns for genetic markers in the LSXSS recombinant-inbred series.

We present the strain distribution patterns (SDPs) of 118 SSLP markers and three pigmentation genes that have been characterized in 27 strains from the LSXSS RI series. This coarse map provides a resource for linkage studies of phenotypes that are heritable in the LSXSS RI series. The LSXSS recombinant inbred (RI) strains were derived from the Long-Sleep (LS) and Short-Sleep (SS) selected lines of mice that were selected for differential sensitivity to ethanol but are also differentially sensitive to a variety of other alcohols, barbiturates, sedative hypnotics, and general anesthetics. Since the parents were not inbred, two atypical factors are present in these SDPs. First, more than two alleles are frequently found in these RIs, and second, some alleles can be uniquely associated with one or the other parent while other alleles may be found in both parental lines. To validate the markers found in the parental line, we genotyped all parental mice from one generation of both the LS and SS lines, thus leading to a set of marker SDPs that are useful for further phenotypic association and identification of provisional QTLs.

Propofol produces differences in behavior but not chloride channel function between selected lines of mice.

We report differential central nervous system (CNS) sensitivity to propofol between Long Sleep (LS) and Short Sleep (SS) mice, selectively bred for their differential CNS sensitivity to ethanol. Intravenous propofol requirements for loss of righting reflex, or sleep time, were measured to define the extent of this sensitivity. LS mice slept approximately two times longer than SS mice at equal doses. Awakening plasma and brain levels of the SS line were, respectively, two and three times that of the LS line (P < 0.0001). This suggests that the LS and SS sleep time difference is CNS mediated, and that propofol and ethanol may share common genes that determine anesthetic sensitivities. The ethanol effect may be at least partially mediated by gamma-aminobutyric acid-A (GABAA) receptor function. Propofol had no differential effect on GABAA receptor function, as measured by chloride flux in LS and SS brain microsac preparations. Either the GABAA receptor does not mediate propofol sleep time, or qualitative differences cannot be demonstrated using 36Cl- uptake in brain membranes.

Genetic models in the study of anesthetic drug action.

This chapter reviews the use of genetic models in the study of anesthetic drug action. Genetic model systems provide a novel approach to understanding mechanisms of anesthetic drug action. Many models have been derived using selection processes that emphasize differential drug sensitivity, producing animal lines that differ in their CNS drug response. Studies of vertebrate (rodent) and invertebrate (Drosophila, Caenorhabditis elegans) animal model systems are covered. The review discusses studies employing lines derived from spontaneous and induced mutagenic processes, selectively bred lines, and inbred lines possessing inherent differential drug sensitivities. The primary focus of included studies is the general anesthetic drugs that are commonly used in the clinical setting. These are drugs such as the inhalational agents (halothane, enflurane, isoflurane, nitrous oxide) and the intravenous induction agents (propofol and diazepam). Rodent lines with differential sensitivity to opiates are also discussed. Finally, an approach to identifying and isolating the genes that control anesthetic sensitivity is discussed in a section on mapping quantitative trait loci (QTL) in recombinant inbred lines.

Coordinated maternal imprinting and paternal genotype determined the ultimate level of hepatic cytochrome P-450 and associated monooxygenase activities.

To understand the pre- and postnatal maternal influence on hepatic drug metabolism later in adult life, we have chosen to study the mouse hybrid (129/sv x LS) offspring derived from contrasting parental lines in a designated maternal environment. Results of these studies revealed that the following: 1) sex of the offspring was the major determinant for cytochrome P-450-specific content and for the activity of aryl hydrocarbon hydroxylase, testosterone 16 alpha-hydroxylase, acetanilide hydroxylase, and NADPH-cytochrome c reductase; 2) maternal factor(s) affected cytochrome P-450-specific content and the activities of acetanilide hydroxylase and aryl hydrocarbon hydroxylase but not of testosterone 15 alpha- and 16 alpha-hydroxylases or NADPH-cytochrome c reductase; 3) superimposed upon sex and maternal determination, paternal genotype significantly contributed to adult hepatic cytochrome P-450-specific content; 4) interactions between sex of the offspring and maternal factor(s) accounted for the development of testosterone 15 alpha- and 16 alpha-hydroxylase activities later in adult life; and 5) with the exception of testosterone 16 alpha-hydroxylase activity, other yet undefined factors, including the paternal genotype, contributed to most of the measured hepatic monooxygenase activities. The mechanism of coordinated maternal imprinting and paternal genotype in determining the levels of hepatic cytochrome P-450 and its associated monooxygenase activities is discussed.

Radiation sensitivity and DNA repair in Caenorhabditis elegans strains with different mean life spans.

The sensitivities to three DNA damaging agents (UV and gamma-radiation, methyl methanesulfonate) were measured in four recombinant inbred (RI) strains of Caenorhabditis elegans with mean life spans ranging from 13 to 30.9 days, as well as in the wild-type strains used to derive these RI's. Sensitivities at several stages in the developmental cycle were tested. There were no significant correlations between mean life span and the lethal effects of these 3 agents. Excision of two UV-radiation-induced DNA photoproducts was also measured. Long-lived strains were no more repair competent than shorter-lived strains. These data indicate that DNA repair plays at best a minor role in the aging process of C. elegans.

Caenorhabditis elegans DNA does not contain 5-methylcytosine at any time during development or aging.

DNA, isolated from age-synchronous senescent populations of Caenorhabditis elegans has been quantitatively and qualitatively analyzed for the presence of 5-methylcytosine. High performance liquid chromatography on two wild-type and several mutant strains of C. elegans failed to detect any 5-methylcytosine. The restriction endonuclease isoschizomers, HpaII and MspI, were used to digest genomic DNA after CsCl purification and failed to detect any 5' cytosine methylation at any age. We conclude that C. elegans does not contain detectable (0.01 mole percent) levels of 5-methylcytosine.

Inducible aldehyde dehydrogenases from rat liver cytosol.

Two rat hepatic cytoplasmic isozymes of aldehyde dehydrogenase, phi, induced by phenobarbital treatment, and tau, induced by TCDD treatment, have been purified from rat hepatic cytosol by ammonium sulfate fractionation, followed by ion-exchange and affinity chromatography. The specific activities of the two isozymes at pH 9.6 with propionaldehyde as substrate and NAD as cofactor were 2850 and 5250 nmol of NADH/min/mg protein for phi and tau isozymes, respectively. Estimates of molecular weights from gel filtration chromatography gave values of 118,000 Da for phi and 106,000 Da for tau. An isoelectric point for the tau enzyme of 6.5 was determined in an electrofocusing column, and approximately 7.2 for phi by immunoelectrophoresis. Both enzymes can oxidize a wide variety of aldehyde substrates, with Km values ranging from millimolar to micromolar. Long-chain aliphatic and aromatic aldehydes using NAD as cofactor tend to be the best utilized substrates. Only the tau enzyme is able to use NADP as cofactor. The measured Km for phi at pH 7.2 for acetaldehyde was 1.97 mM and for tau, 12.1 mM. Both enzymes showed similar inhibition characteristics with sodium arsenite and disulfiram, although the phi enzyme tended to be slightly more sensitive to all inhibitors.