Harrell E. Hurst

Metabolism of lipid peroxidation product, 4-hydroxynonenal (HNE) in rat erythrocytes: role of aldose reductase.

Lipid peroxidation represents a significant source of erythrocyte dysfunction and aging. Because the toxicity of lipid peroxidation appears to be in part due to aldehydic end products, we examined, in rat erythrocytes, the metabolism of 4-hydroxy-trans-2-nonenal (HNE), one of the most abundant and toxic lipid-derived aldehydes. Packed erythrocytes, 0.1 ml, completely metabolized 20 nmoles of HNE in 20 min. The glutathione conjugate of HNE and 4-hydroxynonanoic acid (HNA) represented 70 and 25% of the total metabolism, respectively. Approximately 70% of the metabolites were extruded to the medium. Upon electrospray ionization mass spectrometry, the glutathione conjugate resolved into two distinct species corresponding to glutathionyl HNE (GS-HNE) and glutathionyl 1,4-dihydroxynonene (GS-DHN). The concentration of GS-DHN formed was twice that of GS-HNE. Inhibition of aldose reductase by sorbinil and tolrestat led to a selective decrease in the formation of GS-DHN, although the extent of HNE glutathiolation was unaffected. Inhibitors of aldehyde or alcohol dehydrogenase, i.e., cyanamide and 4-methyl pyrazole, had no effect on the formation of HNA and GS-DHN, indicating that these enzymes are not significant participants in the erythrocyte HNE metabolism. Thus, oxidation to HNA, conjugation with glutathione, and further reduction of the conjugate by aldose reductase appear to be the major pathways of HNE metabolism in erythrocytes. These pathways may be critical determinants of erythrocyte toxicity due to lipid peroxidation-derived aldehydes.

Oral administration of nicotine: Its uptake and distribution after chronic administration to mice

An investigation was made of the suitability of administering nicotine to experimental animals by inclusion in the drinking water. It was found that, after an initial accommodation period of several weeks, nicotine could be administered up to a concentration of 100 micrograms/ml with no decrease in fluid intake or weight gain compared to control. An analysis of the steady-state plasma levels and distribution of nicotine was made in mice which had received nicotine in the drinking water at a concentration of 60 micrograms/ml. The average daily dose of nicotine received by these animals was 17.2 mg/kg. The steady-state plasma level of nicotine was 34.4 ng/ml, representing 6% of the total compound present at steady-state as determined by thin-layer chromatography. The distribution of nicotine or metabolite in mice which had received [methyl-14C]-nicotine orally was determined. Whole-body autoradiography, as well as direct tissue counting, demonstrated that nicotine accumulates in a number of areas, particularly the salivary gland, nasal epithelium, uterus, and liver. There was relatively little material in the blood or brain. This investigation indicates that ad libitum oral administration is an acceptable method for maintaining experimental animals on nicotine for long periods of time.

Early Changes in Gene Expression Induced by Tobacco Smoke: Evidence for the Importance of Estrogen within Lung Tissue

Lung cancer is the leading cause of cancer deaths in the United States, surpassing breast cancer as the primary cause of cancer-related mortality in women. The goal of the present study was to identify early molecular changes in the lung induced by exposure to tobacco smoke and thus identify potential targets for chemoprevention. Female A/J mice were exposed to either tobacco smoke or HEPA-filtered air via a whole-body exposure chamber (6 h/d, 5 d/wk for 3, 8, and 20 weeks). Gene expression profiles of lung tissue from control and smoke-exposed animals were established using a 15K cDNA microarray. Cytochrome P450 1b1, a phase I enzyme involved in both the metabolism of xenobiotics and the 4-hydroxylation of 17β-estradiol (E2), was modulated to the greatest extent following smoke exposure. A panel of 10 genes were found to be differentially expressed in control and smoke-exposed lung tissues at 3, 8, and 20 weeks (P < 0.001). The interaction network of these differentially expressed genes revealed new pathways modulated by short-term smoke exposure, including estrogen metabolism. In addition, E2 was detected within murine lung tissue by gas chromatography-coupled mass spectrometry and immunohistochemistry. Identification of the early molecular events that contribute to lung tumor formation is anticipated to lead to the development of promising targeted chemopreventive therapies. In conclusion, the presence of E2 within lung tissue when combined with the modulation of cytochrome P450 1b1 and other estrogen metabolism genes by tobacco smoke provides novel insight into a possible role for estrogens in lung cancer. Cancer Prev Res; 3(6); 707–17. ©2010 AACR.

Identification and quantitation of N-(carboxymethyl)valine adduct in hemoglobin by gas chromatography/mass spectrometry

A sensitive, specific and reproducible method was developed for the quantitation of the hemoglobin (Hb) adduct N-(carboxymethyl)valine (CMV). This adduct is one of various products from the Maillard reaction, involving reducing sugars and amino acids, proteins or other molecules with a free amino group. Such adducts, including N epsilon-(carboxymethyl)lysine (CML), are called advanced glycation end products (AGE) and have been correlated with aging and severity of diabetes in human tissues. This method was developed to examine the CMV-Hb adduct as a possible AGE formed by reaction of Hb with glucose or other oxidation products. CMV was cleaved selectively from isolated globin using pentafluorophenyl isothiocyanate (PFPITC) in a modified Edman degradation at pH 9.5. The carboxyl group of the adduct was derivatized to its methyl ester with diazomethane. The resulting derivative, 5-isopropyl-1-(methyl acetate)-3-pentafluorophenyl-2-thiohydantoin, was detected by gas chromatography/mass spectrometry with selected ion monitoring (GC/SIM/MS). Quantitation was based on the response factor of the derivative molecular ion (m/z 396) from synthesized CMV and N-(2-carboxyethyl)valine (molecular ion m/z 410) as internal standard. This method exhibits reproducibility and linearity in the range 0.2-100 ng CMV. The limit of quantitation (0.2 ng CMV) gave a signal-to-noise ratio greater than 5:1 using a 1:30 sample aliquot. The GC/SIM/MS method can detect CMV adduct in 5 mg globin samples with relative standard deviations less than 5%. This approach avoids tedious acid hydrolysis and interference from other amino acids. The molecular ion and other CMV derivative ion assignments from samples were confirmed by accurate mass determinations using GC/high resolution SIM/MS. Measurements from random mouse, rat and human globin samples gave mean CMV levels of about 6, 5 and 14 nmol g-1 Hb in these species, respectively.

Follicular fluid Lidocaine levels during transvaginal oocyte retrieval

Lidocaine has been shown to have adverse effects on mouse oocyte fertilization and embryo development. We have demonstrated the presence of pharmacologic levels of lidocaine in human serum and follicular fluid obtained during ultrasound guided transvaginal oocyte retrieval. The significance of this finding is unclear, as four of the eight patients studied became pregnant, including the patient with the highest follicular fluid lidocaine levels. Further evaluation of the effect of lidocaine on human embryos is warranted.

Mood during Epidural Patient-controlled Analgesia with Morphine or Fentanyl

Background Mood states during epidural opioids are not known. The authors studied the change in mood during the 48‐h period of epidural morphine and epidural fentanyl in 47 patients after elective hip or knee joint arthroplasty. Methods An epidural catheter was inserted at the L2‐L3 or L3‐L4 interspace. Anesthesia was induced with thiopenthal and maintained with isoflurane and nitrous oxide. One hour before the conclusion of the operation, patients received an epidural bolus injection of 2 mg morphine (n = 23) or 100 micro gram fentanyl (n = 24), followed by the same opiate (125 micro gram/ml morphine or 25 micro gram/ml fentanyl) epidurally delivered by a patient‐controlled analgesia (PCA) pump in the postoperative period for 48 h. Mood was assessed using the bipolar form of the Profile of Mood States before operation and 24 h, 48 h, and 72 h after operation. Results There was no significant difference in pain intensity between the groups during epidural PCA. Mood states became more positive over time in the patients who received morphine (P < 0.01 at 48 h) and negative in those who were given fentanyl (P < 0.01 at 24 and 48 h, respectively) compared with those before the operation, and they were more positive in the morphine than in the fentanyl group at 24 h, 48 h (P < 0.05), and 72 h (P < 0.01). Patients in the morphine group were more composed, agreeable, elated, confident, energetic, and clearheaded than were those in the fentanyl group (P < 0.05). There was no correlation between mood scores and pain scores in either group. There was an inverse correlation at 48 h between mood scores and plasma fentanyl concentrations (r = ‐0.58, P < 0.05). Conclusion Mood states are significantly more positive during epidural morphine PCA than they are during epidural fentanyl PCA.

Toxicology of 1,3-Butadiene, Chloroprene, and Isoprene

The diene monomers, 1,3-butadiene, chloroprene, and isoprene, respectively, differ only in substitution of a hydrogen, a chlorine, or a methyl group at the second of the four unsaturated carbon atoms in these linear molecules. Literature reviewed in the preceding sections indicates that these chemicals have important uses in synthesis of polymers, which offer significant benefits within modern society. Additionally, studies document that these monomers can increase the tumor formation rate in various organs of rats and mice during chronic cancer bioassays. The extent of tumor formation versus animal exposure to these monomers varies significantly across species, as well among strains within species. These studies approach, but do not resolve, important questions of human risk from inhalation exposure. Each of these diene monomers can be activated to electrophilic epoxide metabolites through microsomal oxidation reactions in mammals. These epoxide metabolites are genotoxic through reactions with nucleic acids. Some of these reactions cause mutations and subsequent cancers, as noted in animal experiments. Significant differences exist among the compounds, particularly in the extent of formation of highly mutagenic diepoxide metabolites, when animals are exposed. These metabolites are detoxified through hydrolysis by epoxide hydrolase enzymes and through conjugation with glutathione with the aid of glutathione S-transferase. Different strains and species perform these reactions with varying efficacy. Mice produce these electrophilic epoxides more rapidly and appear to have less adequate detoxification mechanisms than rats or humans. The weight of evidence from many studies suggests that the balance of activation versus detoxification offers explanation of differing sensitivities of animals to these carcinogenic actions. Other aspects, including molecular biology of the many processes that lead through specific mutations to cancer, are yet to be understood. Melnick and Sills (2001) compared the carcinogenic potentials of these three dienes, along with that of ethylene oxide, which also acts through an epoxide intermediate. From the number of tissue sites where experimental animal tumors were detected, butadiene offers greatest potential for carcinogenicity of these dienes. Chloroprene and then isoprene appear to follow in this order. Comparisons among these chemicals based on responses to external exposures are complicated by differences among studies and of species and tissue susceptibilities. Physiologically based pharmacokinetic models offer promise to overcome these impediments to interpretation. Mechanistic studies at the molecular level offer promise for understanding the relationships among electrophilic metabolites and vital genetic components. Significant improvements in minimization of industrial worker exposures to carcinogenic chemicals have been accomplished after realization that vinyl chloride caused hepatic angiosarcoma in polymer production workers (Creech and Johnson 1974; Falk et al. 1974). Efforts continue to minimize disease, particularly cancer, from exposures to chemicals such as these dienes. Industry has responded to significant challenges that affect the health of workers through efforts that minimize plant exposures and by sponsorship of research, including animal and epidemiological studies. Governmental agencies provide oversight and have developed facilities that accomplish studies of continuing scientific excellence. These entities grapple with differences in perspective, objectives, and interpretation as synthesis of knowledge develops through mutual work. A major challenge remains, however, in assessment of significance of environmental human exposures to these dienes. Such exposure levels are orders of magnitude less than exposures studied in experimental or epidemiological settings, but exposures may persist much longer and may involve unknown but potentially significant sensitivities in the general population. New paradigms likely will be needed for toxicological evaluation of these human exposures, which are ongoing but as yet are not interpreted.

Midazolam attenuates ketamine-induced abnormal perception and thought process but not mood changes.

Purpose: To determine the effects of midazolam, 30 ng·mL−1, on altered perception, mood, and cognition induced by ketamine.Methods: After ketamine was administered to achieve target concentrations of 50, 100, or 150 ng·mL−1 in 11 volunteers, perception, mood, and thought process were assessed by a visual analog scale. Mini-Mental State examination (MMSE) assessed cognition. Boluses of midazolam, 30, 14.5, and 12µg·kg−1, were injected every 30 min to maintain the plasma concentration at 30 ng·mL−1, which was reached 30 min after each injection.Results: Ketamine produced changes in perception about the body (P<0.01, 0.001, and 0.001 at 30, 60, and 90 min), surroundings (P<0.01 and 0.0001 at 60 and 90 min), time (P<0.002 and 0.0001 at 60 and 90 min), reality (P<0.001 and 0.0001 at 60 and 90 min), sounds (P<0.002 at 90 min), and meaning (P<0.05 at 90 min). Subjects felt less energetic and clearheaded (P<0.02 and 0.05) during ketamine, midazolam, and their co-administration. Ketamine impaired thought process (P<0.003 and 0.0001 and 60 and 90 min). Ketamine and midazolam decreased mean total MMSE and recall scores (P<0.001 for both). Co-administraion reduced the number of subjects with perceptual (body,P<0.01 and 0.001 at 30 and 60 min) and thought process abnormalities. Within the range of observation, co-administration did not affect the changes in mood or recall.Conclusion: Midazolam attenuates ketamine-induced changes in perception and thought process.RésuméObjectif: Déterminer les effets de 30 ng·mL−1 de midazolam sur les changements de perception, d’humeur et de fonction cognitive induits par la kétamine.Méthode: Après l’administration de kétamine visant à obtenir des concentrations cibles de 50, 100, ou 150 ng·mL−1 chez 11 volontaires, la perception, l’humeur et la fonction cognitive ont été évaluées à l’aide d’une échelle visuelle analogique. L’examen MMS de Folstein et coll. (MMS) a servi à évaluer la fonction cognitive. Des bolus de midazolam de 30, 14,5 et 12µg·kg−1 ont été injectés à toutes les 30 min afin de maintenir la concentration plasmatique à 30 ng·mL−1, concentration atteinte 30 min après chaques injection.Résultats: La kétamine a modifié la perception du corps (P<0,01; 0,001 et 0,0001 à 30, 60 et 90 min), de l’environnement (P<0,01 et 0,0001 à 60 et 90 min), du temps (P<0,002 et 0,0001 à 60 et 90 min), de la réalité (P<0,001 et 0,0001 à 60 et 90 min), des sons (P <0,002 à 90 min) et du sens (P<0,05 à 90 min). Les sujets se sentaient moins énergiques et moins lucides (P<0,02 et 0,05) pendant l’administration de kétamine, de midazolam et pendant leur co-administration. La kétamine a altéré le processus cognitif (P<0,003 et 0,0001 à 60 et 90 min). La kétamine et le midazolam ont fait baisser les scores totaux moyens de MMS et de mémoire (P<0,001 pour les deux). La co-administration a réduit le nombre de sujets dont les perceptions (corps,P<0,01 et 0,001 à 30 et 60 min) et la fonction cognitive étaient modifiées. Pendant le temps d’observation, la co-administration n’a pas eu d’effet sur les changements d’humeur ou de mémoire.Conclusion: Le midazolam diminue les changements de perception et de cognition induits par la kétamine.

Doxapram only slightly reduces the shivering threshold in healthy volunteers

We determined the effects of doxapram on the major autonomic thermoregulatory responses in humans. Nine healthy volunteers were studied on 2 days: control and doxapram (IV infusion to a plasma concentration of 2.4 ± 0.8, 2.5 ± 0.9, and 2.6 ± 1.1 &mgr;g/mL at the sweating, vasoconstriction, and shivering thresholds, respectively). Each day, skin and core temperatures were increased to provoke sweating, then reduced to elicit peripheral vasoconstriction and shivering. We determined the sweating, vasoconstriction, and shivering thresholds with compensation for changes in skin temperature. Data were analyzed with paired t-tests and presented as mean ± sd; P < 0.05 was considered statistically significant. Doxapram did not change the sweating (control: 37.5° ± 0.4°C, doxapram: 37.3° ± 0.4°C; P = 0.290) or the vasoconstriction threshold (36.8° ± 0.7°C versus 36.4° ± 0.5°C; P = 0.110). However, it significantly reduced the shivering threshold from 36.2° ± 0.5°C to 35.7° ± 0.7°C (P = 0.012). No sedation or symptoms of panic were observed on either study day. The observed reduction in the shivering threshold explains the drug’s efficacy for treatment of postoperative shivering; however, a reduction of only 0.5°C is unlikely to markedly facilitate induction of therapeutic hypothermia as a sole drug.

Quantification of amitriptyline, nortriptyline, and 10-hydroxy metabolite isomers in plasma by capillary gas chromatography with nitrogen-sensitive detection

A selective, sensitive method for the determination of amitriptyline and its metabolites is described. This method involves liquid-liquid extraction and capillary gas chromatography with nitrogen-sensitive detection. The detection limits of amitriptyline, nortriptyline, 10-hydroxy(E)amitriptyline, 10-hydroxy(E)nortriptyline, and 10-hydroxy(Z)nortriptyline were slightly less than 0.5 ng/ml in 1.0-ml plasma samples. The coefficients of variation for within-run and between-run analyses of samples containing 100 ng/ml were less than 12% and 9%, respectively. The method offers rapid analysis of individual isomers, increased sensitivity over high-performance liquid chromatographic methodology and the conveniences of the gas chromatographic technique.