Genotype differences in cognitive functioning in Noonan syndrome.

Noonan syndrome (NS) is an autosomal-dominant genetic disorder associated with highly variable features, including heart disease, short stature, minor facial anomalies and learning disabilities. Recent gene discoveries have laid the groundwork for exploring whether variability in the NS phenotype is related to differences at the genetic level. In this study, we examine the influence of both genotype and nongenotypic factors on cognitive functioning. Data are presented from 65 individuals with NS (ages 4-18) who were evaluated using standardized measures of intellectual functioning. The cohort included 33 individuals with PTPN11 mutations, 6 individuals with SOS1 mutations, 1 individual with a BRAF mutation and 25 participants with negative, incomplete or no genetic testing. Results indicate that genotype differences may account for some of the variation in cognitive ability in NS. Whereas cognitive impairments were common among individuals with PTPN11 mutations and those with unknown mutations, all of the individuals with SOS1 mutations exhibited verbal and nonverbal cognitive skills in the average range or higher. Participants with N308D and N308S mutations in PTPN11 also showed no (or mild) cognitive delays. Additional influences such as hearing loss, motor dexterity and parental education levels accounted for significant variability in cognitive outcomes. Severity of cardiac disease was not related to cognitive functioning. Our results suggest that some NS-causing mutations have a more marked impact on cognitive skills than others.

Caffeine can affect velocity in the middle cerebral artery during hyperventilation, hypoventilation, and thinking: a transcranial Doppler study.

This study examined possible caffeine-mediated changes in blood flow velocity in the middle cerebral artery (VMCA) induced by tests of cerebrovascular responsiveness. Transcranial Doppler (TCD) sonography provided simultaneous bilateral VMCA measures while healthy college students hypoventilated, hyperventilated, and performed cognitive activities (short-term remembering, generating an autobiographical image, solving problems), each in 31-second tests. VMCA measures were obtained from the same persons, in separate testing sessions, when they were noncaffeinated and under two levels of caffeine: a smaller amount (from a cola, 45 mg/12 oz) and a larger amount (from coffee, 117 mg/8 oz). Compared with the no-caffeine control condition, a smaller amount of caffeine had no significant effects on global VMCA, but a larger amount suppressed VMCA by 5.8%. Time-course analyses showed that VMCA (1) followed a triphasic pattern to increase over baselines during hypoventilation regardless of caffeine condition, (2) slowed below baselines during hyperventilation (with the degree of slowing attenuated under caffeine), and (3) increased over baselines during all cognitive activities (ranges 3.8-6.9%). It is concluded that a large amount of caffeine can suppress VMCA, and this possibility should be anticipated when TCD is used to assess cerebral hemovelocity.

How medical students can bring about curricular change.

Traditionally, medical school committees have been charged with curricular improvement and modification, while medical students have had little or no involvement in reform efforts. However, medical students can sometimes be ahead of faculty in recognizing new topics that need to be covered, and their energy, commitment, and vision can be a very important impetus for curricular change. In 1995-96, as part of a general curricular restructuring effort, faculty at Dartmouth Medical School began to design and offer new electives in innovative topics, with the idea that electives might become part of the required curriculum if the material presented in them were deemed to be "core." Students were invited to organize their own electives if a topic in which they were interested was not being covered. The authors (two were second-year medical students and the third was their faculty sponsor) developed an elective in women's health. This paper describes the development and implementation of this elective, and the process by which the course was later made part of the required curriculum at Dartmouth. The success of the authors' efforts highlights the crucial role students can play in reforming medical curricula.

Blood flow velocities in three cerebral arteries in the same subjects modulate during thinking.

College students (n = 22) engaged in thinking activities while simultaneous bilateral velocity (V) measures are obtained from their middle, anterior, and posterior cerebral arteries (MCA, ACA, and PCA). The study follows a 3 x 2 x 6 factorial design with repeated measures on artery insonated (MCA, ACA, and PCA), hemisphere (right and left), and six experimental phases, within which an initial 62-second baseline (BL) period is followed by five 31-second thinking tasks (short-term remembering, generating an image, making decisions, and solving language and math problems). VMCA is faster than BL during each thinking task (range, 4.9%-8.5%; p < 0.001), but changes in VACA, VPCA, and all hemispheric differences are not significant. A stronger degree of increase is present for VMCA than (1) both VACA and VPCA during short-duration remembering, making decisions, and working math problems, and (2) VACA, which in turn is stronger than VPCA when generating images and constructing new words. The authors' study shows that transcranial Doppler neuroimaging conveniently provides physiological indices of thinking from three cerebral arteries of the same subjects.

Modulations in cerebral hemodynamics under three response requirements while solving language-based problems: a transcranial Doppler study.

This experiment used transcranial Doppler ultrasound to measure blood flow velocity in the middle, anterior or posterior cerebral artery (MCA, ACA and PCA, respectively) while separate groups of college students (each n = 20) solved anagrams and constructed new words using letters of a target word, each while viewing, speaking or writing the responses. The silent viewing requirement affected global velocities only while constructing words: velocities in both the MCA and ACA were faster than in the PCA. Speaking the solutions during both types of problems yielded faster overall velocities in the MCA than in the PCA. Finally, writing the solutions while constructing words led to faster velocities in the MCA compared to both the ACA and PCA. Time-course patterns to velocity changes from the thinking periods showed elevations in MCA velocity at the beginning and end of the periods, while PCA velocity typically slowed below baseline in the middle of the periods. These data show that the kind of language-based problem-solving task and the specific response requirement arranged to accomplish a task selectively affected velocity in three cerebral arteries.

Induction of liver cytochrome P-450 (CYP) 3A in male and female rats by a steroidal androgen receptor antagonist, zanoterone.

The cytochrome P-450 (CYP) mediated hydroxylation of testosterone to 6 beta-, 7 alpha-, and 16 alpha-hydroxytestosterone (b beta-, 7 alpha-, and 16 alpha-OHT) and the dealkylation of ethoxycoumarin to 7-hydroxycoumarin (ECOD) and ethoxyresorufin to resorufin (EROD) were used to probe changes in CYP monooxygenase activities in liver microsomes from rats treated with the androgen receptor antagonist, zanoterone (Z). Phenobarbital (PB) and beta-naphthoflavone (beta-NF) were used as comparators. There were sex-related differences in the constitutive CYP activities and in the responses of CYP activities to Z. The greatest effect of Z administration was on 6 beta-OHT activity: It was increased up to 5.2-fold in males and 13.9-fold in females (Z high dose). The effect was larger than the produced by PB or beta-NF (< or = threefold increases). Z (high dose), PB, and beta-NF increased ECOD to a similar extent, e.g., about 1.3-fold in males and 1.2-2.9-fold in females. beta-NF increased EROD (11.2-fold males, 6.2-fold females) more than PB (3.4- to 4.6-fold) or Z (1.3- to 1.7-fold). Since hydroxylation of testosterone at the 6 beta position in rats and humans is catalyzed primarily by CYP isoforms from the 3A subfamily, the increase in 6 beta-OHT suggests that Z induced CYP 3A activity. These findings were confirmed with Western immunoblots with probes for rat CYP 1A1, 2B1/2, 2E1, 3A, and 4A. Z produced a three-to fourfold increase in the 3A isoform for both male and female rats. Results from this study suggest that in a clinical setting, Z therapy has the potential to induce CYPs of the 3A subfamily and in so doing alter the metabolism and clearance of drugs that are substrates for the 3A subfamily.

Dose-dependent urinary excretion of acrylonitrile metabolites by rats and mice.

The dose dependence of the urinary excretion of acrylonitrile (ACN) metabolites was studied after oral administration of [2,3-14C]ACN to male F-344 rats (0.09 to 28.8 mg/kg) and male B6C3F1 mice (0.09 to 10.0 mg/kg). Urine was the major route of excretion of ACN metabolites (77 to 104% of the dose), with less than 8% of the dose excreted in the feces. Reverse-phase HPLC analysis of urine from treated animals indicated five major components (1 through 5 in order of elution) that accounted for 75 to 100% of the total urinary radioactivity. Component 4 was observed in the urine of ACN-treated mice but was only present in trace amounts in the urine of ACN-treated rats. Components 1, 2, and 3 were present in the urine of animals administered [2,3-14C]cyanoethylene oxide (CEO), indicating that these components were derived from the epoxide metabolite of ACN. The ACN urinary metabolites were isolated by HPLC and identified by chromatographic and mass spectral analysis. Component 5 was N-acetyl-S-(2-cyanoethyl)cysteine and component 4 was S-(2-cyanoethyl)thioacetic acid, both derived from the glutathione (GSH) conjugate of ACN. Component 3 contained N-acetyl-S-(2-hydroxyethyl)cysteine, N-acetyl-S-(carboxymethyl)cysteine, and N-acetyl-S-(1-cyano-2-hydroxyethyl)cysteine. Component 2 was thiodiglycolic acid. These urinary metabolites are derived from catabolism of the GSH conjugates of CEO. The polar component 1 was not identified. These results demonstrate that GSH conjugation is the major disposition pathway of ACN. The excretion of metabolites derived from CEO was an approximately linear function of dose in both species, whereas the excretion of N-acetyl-S-(2-cyanoethyl)cysteine increased nonlinearly with dose. This nonlinearity indicates the presence of a saturable pathway competing with glutathione for ACN, most likely the cytochrome P450-dependent oxidation of ACN. Thiodiglycolic acid was formed 10-fold more in mice than in rats, but this species difference in the oxidative processing of GSH conjugates is probably not of toxicological significance. The ratio of ACN epoxidation to GSH conjugation was 0.50 in rats and 0.67 in mice. This species difference in ACN oxidation could have important toxicological implications, since CEO is believed to mediate the carcinogenic effects of ACN.

Species comparison of acrylonitrile epoxidation by microsomes from mice, rats and humans: relationship to epoxide concentrations in mouse and rat blood.

Acrylonitrile (ACN) has been shown to cause tumors of the brain, stomach and Zymbal's gland in rats in several bioassays, but it has not been tested in other species. The carcinogenic risk of humans exposed to ACN is unclear. ACN is metabolized in the liver to 2-cyanoethylene oxide (CEO), which is believed to be the proximate or ultimate carcinogenic species. Therefore, the kinetics of CEO formation were studied with liver and lung microsomes from mice and humans using a GC-MS assay for CEO, and the data were compared with previously obtained kinetic parameters for rat microsomal enzymes. The rate of CEO formation by human liver microsomes was comparable to that of rat liver microsomes, but less than that of mouse liver microsomes. Liver microsomes produced more CEO than lung microsomes with all three species. CEO formation by microsomes from mice was approximately 4 times greater than that by microsomes from rats or humans, suggesting that mice would have higher CEO concentrations in blood than rats after ACN exposure. However, after oral administration of ACN, the concentration of CEO in mouse blood was one-third that in rat blood at all doses and time points examined. These results show that CEO circulates via the blood, providing exposure to distant sites. The blood concentrations of CEO do not appear to correlate with rates of microsomal CEO formation. This suggests that species differences in the detoxication of CEO may play an important role in determining circulating CEO concentrations and distant organ exposure.

Socioeconomic status, ethnicity, psychological distress, and readiness to utilize a mental health facility.

The effects of minority status versus ethnic culture on Mexican-Americans' underutilization of mental health services were reassessed through development and testing of an analytic path model that proposes a sequence of factors, including Mexican-American ethnicity, socioeconomic status, degree of social and institutional support, and depression, which culminate in a person's decision to utilize mental health facilities. The model also predicts that life stress will affect utilization through its influence on depression. Data from 783 subjects generally supported the model's predictions. A multifactorial approach to the causes of mental health problems and utilization behavior in the Mexican-American population is suggested.

The effect of m-xylene on rat lung benzo[a]pyrene metabolism and microsomal membrane lipids: comparison with p-xylene.

m-Xylene (1 g/kg, i.p., 1 h) was shown to decrease aryl hydrocarbon hydroxylase (AHH) activity, a detoxification pathway for benzo[a]pyrene (BaP), in the rat lung. Inhibition was maximal at 1 g/kg, 1 h after treatment and was sustained for at least 24 h. Reduction in cytochrome P-450 activity in rat lung was also observed, while liver activity was unchanged. p-Xylene has been previously shown to produce a similar pattern of MFO changes in rat lung. The lipid composition of the microsomal membrane is important to mixed function oxidase (MFO) regulation and function. Since the xylenes are lipophilic, these compounds were studied to determine whether they alter pulmonary microsomal lipids. p-Xylene produced an organ specific increase in lipid peroxidation in the rat lung. This was accompanied by decreases in lung microsomal total phospholipid (PL) and phosphatidylcholine (PC) content. Pulmonary microsomal membrane fluidity was also reduced by p-xylene administration. In comparison, m-xylene administration did not change any of the lipid membrane parameters tested. These divergent results leave unresolved the role of altered PL metabolism in solvent-induced inhibition of MFO activity.

Metabolism of acrylonitrile to 2-cyanoethylene oxide in F-344 rat liver microsomes, lung microsomes, and lung cells.

The metabolism of acrylonitrile to the epoxide, 2-cyanoethylene oxide (ANO) was examined in rat liver microsomes, lung microsomes, and isolated enriched lung cell preparations. GC/high resolution MS was used to quantitate ANO in microsomal and cellular extracts by monitoring the fragment ion C2H3N (m/z 41.0265). The limit of detection was 0.05 pmol of ANO/0.5 microliter of standard solution, microsomal extract, or cellular extract injected onto the column, and the linear range of analysis was 0.05 to 12.5 pmol of ANO. Kinetic parameters of Vmax, V/K, and Km were calculated for microsomal ANO formation. Liver microsomes were quantitatively more active than lung microsomes on a mg of protein basis. The Vmax (pmol of ANO formed/min/mg of protein) was 666.61 for liver and 45.07 for lung microsomes. The V/K (pmol of ANO/min/mg of protein/microM) was 12.83 for liver and 0.02 for lung microsomes. The apparent Km was 51.93 microM and 1853.83 microM for liver and lung microsomes, respectively. When calculated as nmol of ANO formed/min/nmol of microsomal P-450, the Vmax for lung was equivalent to the Vmax for liver. ANO formation in the rat lung was cell specific. The rates of metabolism in the Clara cell-enriched fraction, the alveolar type II cell-enriched fraction, and the cell suspension were 2.55, 0.38, and 0.67 pmol of ANO formed/min/mg of protein, respectively. No metabolism was observed in the endothelial (small) cell-enriched fraction or in the alveolar macrophages. The results suggest that the lung contributes to the metabolism and disposition of inhaled acrylonitrile.

Effect of glutathione depletion on the irreversible association of acrylonitrile with tissue macromolecules after oral administration to rats.

Binding of acrylonitrile and its reactive metabolites to tissue macromolecules, especially nucleic acids, may be responsible for its carcinogenicity in rats. Both acrylonitrile and its primary metabolite, 2-cyanoethylene oxide, also react with glutathione. To better understand the role of glutathione in the manifestation of acrylonitrile toxicity, the irreversible binding to tissue macromolecules was assessed in control and glutathione-depleted F-344 rats treated with a 4 mg/kg dose (po) of [2,3-14C]acrylonitrile. Glutathione was depleted in rat tissues by the administration of a combined intraperitoneal phorone/buthionine sulfoximine treatment (300 mg/kg and 2 mmol/kg, respectively) given 30 min prior to acrylonitrile administration. The amount of total radioactivity recovered from brain, stomach (target organs), liver, kidney, lung, and blood (nontarget organs) was similar between control and glutathione-depleted rats. However, stomach, lung, blood, and liver showed an increase in total radioactivity content after glutathione depletion by phorone/buthionine sulfoximine treatment. Glutathione depletion also caused an increase in acrylonitrile-derived non-dialysable radioactivity (MW greater than 3500 Da) in liver, lung, kidney, stomach, blood, and brain macromolecules between 6 and 24 hr after the dose. There was no organ-specific accumulation of radiolabel in RNA in control rats. However, an increase in the radiolabel associated with nucleic acids in the target organs but not in the nontarget organs was measured in glutathione-depleted rats. Urinary excretion of thiocyanate, a metabolite derived from the epoxide pathway, was also increased by 300% in glutathione-depleted rats. These results suggest that glutathione might play a role in the extent of 2-cyanoethylene oxide formation and in the distribution of the radiolabel among tissues.

Effect of glutathione depletion on the uptake of acrylonitrile vapors and on its irreversible association with tissue macromolecules.

Cellular GSH may influence the metabolism of the rodent brain and forestomach carcinogen acrylonitrile (ACN) and its subsequent binding to tissue macromolecules. To investigate the role of GSH in ACN metabolism and binding to macromolecules, we studied the effect of GSH depletion on the irreversible association of radiolabel with tissue macromolecules in male F-344 rats given a 4 mg/kg dose of [2,3-14C]ACN by inhalation. A combined phorone/buthionine sulfoximine treatment (300 mg/kg and 2 mmol/kg, respectively) was given 30 minutes prior to ACN exposure to deplete GSH. The uptake of ACN vapor by control rats was biphasic and characterized by a rapid phase lasting about 60 min and by a slower phase from 60 min to the end of exposure. The rate of uptake for both phases was linearly related to the initial concentration of ACN in the chamber. GSH depletion caused an increase in the rate of ACN uptake in both phases. It also caused a decrease in total radioactivity recovered in brain, stomach, liver, kidney, and blood and a concomitant decrease in the ACN-derived nondialyzable radioactivity in these organs. In control rats, accumulation of radiolabel was greatest in brain RNA, but no radioactivity was detected in DNA of any organ examined. In GSH-depleted rats, the radiolabel concentration was higher in brain RNA than in the liver or stomach RNA, but was also 50% lower than that observed in brain RNA of control rats. Urinary excretion of thiocyanate (SCN-), a metabolite derived from the epoxide pathway of ACN metabolism, was doubled in GSH-depleted rats. These results suggest that GSH might be involved in the distribution of ACN-derived reactive species and, therefore, might play a role in the binding of ACN-derived species to tissue macromolecules and nucleic acids.

Changes in rat lung microsomal lipids after p-xylene: relationship to inhibition of benzo[a]pyrene metabolism.

The relationship between p-xylene's effects on microsomal membranes, cytochrome P-450, and benzo[a]pyrene (BaP) metabolism was studied. p-Xylene (1 g/kg, ip, 1 h) inhibited 3-hydroxy BaP (3-OH) formation and decreased arylhydrocarbon hydroxylase (AHH) activity approximately 40% in rat lung microsomes. BaP dihydrodiol and quinone formation were unchanged by p-xylene administration. Cytochrome P-450 was below the limit of detection in lung microsomes from p-xylene-treated rats. Total phospholipid (PL) and phosphatidylcholine (PC) in microsomal membranes were decreased 28% and 17%, respectively. Cholesterol (CL), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and sphingomyelin (SM) were unchanged. The net activity of enzymes involved in the synthesis of PC, phosphatidylethanolamine-N-methyltransferase I and II (PMT I and PMT II), was slightly elevated by p-xylene. PL/CL and PC/PE ratios, indicators of membrane fluidity, were decreased 34% and 13%, respectively, in microsomes from p-xylene-treated rats. Analysis of fluidity by fluorescence polarization showed that the actual fluidity of treated microsomes was slightly decreased (5%) as compared to controls. The decrease in P-450, PL, and PC is considered to contribute to the inhibition of BaP metabolism.

Regioselective inhibition of benzo(a)pyrene metabolism by corticosterone in comparison with metyrapone and alpha-naphthoflavone.

Corticosterone was investigated for its ability to inhibit benzo(a)pyrene (BaP) metabolism and was compared to metyrapone and alpha-naphthoflavone. Corticosterone inhibited aryl hydrocarbon hydroxylase (AHH) activity nonlinearly in hepatic microsomes from uninduced, phenobarbital-induced, or 3-methylcholanthrene-induced rats. When compared to the classic inhibitors metyrapone and alpha-naphthoflavone, corticosterone had inhibitory properties similar to each. Metabolism of BaP to dihydrodiols was inhibited at the K-region by corticosterone only in uninduced microsomes. Dihydrodiol formation at the Bay region, which leads to the putative ultimate carcinogen, was not affected by corticosterone in uninduced or phenobarbital-induced microsomes but stimulated in 3-methylcholanthrene-induced microsomes. These findings suggest that corticosterone regioselectively inhibits cytochrome P-450 mediated oxidation of BaP to less mutagenic metabolites while stimulating the formation of highly mutagenic products.

Lung metabolism of benzo[a]pyrene in rats treated with p-xylene and/or ethanol.

The metabolism of benzo[a]pyrene (BaP) may be altered by xenobiotic compounds. The effects of p-xylene and ethanol on the lung metabolism of BaP were studied. p-Xylene was administered by ip injection at doses ranging from 0.1 to 1.0 g/kg (1:1 in soybean oil). Ethanol was administered po at 5 g/kg (40% w/v). Rats given p-xylene, ethanol, or p-xylene and ethanol were sacrificed 1 h after treatment. Additional time points of 15 min, 30 min, 4 h, and 24 h after p-xylene (1 g/kg) were examined. 3-Hydroxy-BaP (3-OH) formation was measured fluorometrically as aryl hydrocarbon hydroxylase activity (AHH) in lung microsomes. p-Xylene (1 g/kg) inhibited the formation of 3-OH BaP 40% at 15 min, 27% at 30 min, 43% at 1 h, and 39% at 4 h after treatment. Inhibition of AHH activity was still present 24 h after dosing (41%). AHH activity was inhibited 27% and 46% at 0.5 mg/kg and 1.0 mg/kg p-xylene (1 h), respectively, while the lowest dose (0.1 mg/kg) did not change activity. Analysis of the major metabolites of BaP by high-performance liquid chromatography (HPLC) demonstrated that the formation of 3-OH and 4,5-diol BaP were inhibited 32% and 50%, respectively, in lung microsomes prepared 24 h after a single injection of p-xylene (1 g/kg). None of the other metabolites analyzed were changed by p-xylene. Ethanol had no effect on 3-OH BaP formation during a 1-h treatment. A combined dose of ethanol and p-xylene moderately inhibited 3-OH BaP formation. These findings indicate that BaP detoxication (i.e., 3-OH formation) in rat lung is selectively inhibited by p-xylene but not ethanol. Ethanol appears to modify the inhibitory effect of p-xylene.