9 -Tetrahydrocannabinol and ethyl alcohol: evidence for cross-tolerance in the rat.

Rats trained in a one-way avoidance situlation were made tolerant to the depressant effects of Delta(9)-tetrahydrocannabinol. Ethyl alcohol (3.2 grams per kilogram, intraperitoneally) did not greatly affect rats that were tolerant to delta(9)tetrahydrocannabinol but depressed the behavior of nontolerant rats. Rats made tolerant to ethyl alcohol were less affected by Delta(9)-tetrahydrocannabinol.

Inhibition of arachidonic acid incorporation into erythrocyte phospholipids by peracetic acid and other peroxides. Role of arachidonoyl-CoA: 1-palmitoyl-sn-glycero-3-phosphocholine acyl transferase.

To explore possible mechanisms of the arachidonic acid deficiency of the red blood cell membrane in alcoholics, we compared the effect of ethanol and its oxidized products, acetaldehyde and peracetic acid, with other peroxides on the accumulation of [14C]arachidonate into RBC membrane lipids in vitro. Incubation of erythrocytes with 50 mM ethanol or 3 mM acetaldehyde had no effect on arachidonate incorporation. Pretreatment of erythrocytes with 10 mM hydrogen peroxide, 0.1 mM cumene hydroperoxide or 0.1 mM t-butyl hydroperoxide had little effect on [14C]arachidonate incorporation in the absence of azide. However, pretreatment of cells with N-ethylmaleimide, 0.1 mM peracetic acid or performic acid, with or without azide, inhibited arachidonate incorporation into phospholipids but not neutral lipids. In chase experiments, peracetate also inhibited transfer of arachidonate from neutral lipids to phospholipids. To investigate a possible site of this inhibition of arachidonate transfer into phospholipids by percarboxylic acids, we assayed a repair enzyme, arachidonoyl CoA: 1-palmitoyl-sn-glycero-3-phosphocholine acyl transferase (EC 2.3.1.23). As in intact cells, phospholipid biosynthesis was inhibited more by N-ethylmalemide and peracetic acid than by hydrogen peroxide, cumene hydroperoxide, and t-butyl hydroperoxide. Peracetic acid was the only active inhibitor among ethanol and its oxidized products studied and may deserve further examination in ethanol toxicity.

Organic peroxides inhibit neutrophil leukotriene B4 biosynthesis.

Leukotriene B4, an autacoid metabolite of arachidonic acid produced by polymorphonuclear neutrophils, induces chemokinesis, chemotaxis, and adhesion of these cells at sites of inflammation. Because neutrophil infiltration is a self-limited process, we hypothesized that oxidized lipid products of neutrophil-damaged tissue might inhibit leukotriene B4 biosynthesis, thereby preventing additional neutrophil infiltration and limiting peroxidative tissue damage. Erythrocyte ghosts exposed to a hydrogen peroxide-generating system served as a model of peroxidized tissue in inflammation and inhibited neutrophil leukotriene B4 production by 50% compared with unoxidized ghosts. Organic peroxides, including tert-butylhydroperoxide, peracetic acid, and linoleic hydroperoxide, resembling the product(s) of tissue membrane peroxidation in lipid solubility and catalase resistance, inhibited leukotriene B4 biosynthesis in a dose-dependent manner (50% inhibitory concentration of 3.9 microM compared to 530 microM for H2O2). Biosynthetic steps prior to the 5-lipoxygenase did not appear to be the site of inhibition. Likewise, the step after the 5-lipoxygenase, the leukotriene A4 hydrolase, was not primarily involved. Thus a possible mechanism for controlling the influx of neutrophils and their oxidative damage during inflammation may be inhibition of the 5-lipoxygenase by catalase-resistant lipid peroxides released by tissue membranes.

Assessment of the effectiveness of animal developmental toxicity testing for human safety.

Evaluations of studies for four well-known human developmental toxicants clearly suggest that a margin of exposure of 1/100th the NOAEL for the most sensitive animal species tested provides adequate safety for the human conceptus. The lowest reported human teratogenic exposures occurred at doses at least one log above the estimated "safe" or acceptable daily exposure based on the most sensitive animal species, that is, 1/100th animal NOAEL. (The MOE ranged from < 1 to 10.). The data and analyses are consistent with the conclusion that, regardless of the type of in utero effect produced in animals, the margin of safety of 100 is likely to protect the human conceptus in utero from developmental perturbation, and it is a scientifically reasonable and conservative default number.

Multiple chemical exposures: synergism vs. individual exposure levels.

Exposure to single chemicals is known to produce congenital malformations in both pregnant animals and humans exposed at sufficiently high intensity. However, real life involves multiple, simultaneous exposures. Using as a database the 43 multiple chemical exposure studies located by Nelson (Teratology 49:33-71; 1994) where synergism was reported, we explored the degree to which such concerns may be realistic from the viewpoint of the current standard developmental toxicity safety evaluation process. Focusing on the assessment of the lowest tested dose of a given agent participating in synergistic activity as compared to its threshold level for eliciting toxicity when administered alone, we found that while the availability of adequate data was limited, all cases, with the possible exception of one, demonstrated synergistic toxic expression only when at least one, and usually both, compounds were used at or above their individual threshold for toxicity. These findings suggest that in animals such phenomena of synergistic chemical interactions are likely to occur only when at least one and more likely both agents are administered at or above their individual threshold for toxicity. To the extent animal studies are predictive of human developmental hazards due to single chemical exposures, available data do not establish multiple chemical exposures as a major human developmental concern.

HIV seroprevalence among military blood donors in Manica Province, Mozambique.

HIV seroprevalence data show an alarming HIV situation in central Mozambique, but little is known about the situation of HIV in Mozambican military personnel. This study is a retrospective analysis of laboratory records for voluntary blood donors at a rural hospital from January 1997 through December 1999. The hospital screened blood samples with HIV SPOT rapid test for HIV and rapid plasma reagin (RPR) serological test for syphilis. Of the 797 blood donors during this period, 110 (13.8%) were military personnel of whom 39.1% were HIV positive (35.0% in 1997, 33.3% in 1998 and 48.7% in 1999). Among the 687 nonmilitary donors 15.3% were HIV positive (P<0.0001 vs military). 74.4% of HIV-positive military personnel were also RPR positive. Conversely, only 3.0% of HIV-negative military donors were RPR positive. In light of the high rates of HIV and syphilis in military personnel, aggressive intervention measures must be taken to prevent and treat HIV and STDs in this population.

Fetal development in the normal thick-tailed bushbaby (Galago crassicaudatus panganiensis).

Fetal development was examined at days 60, 75, 90, 105, 120, or 135 of gestation in 19 thick-tailed bushbaby fetuses (Galago crassicaudatus panganiensis) from 17 normal, timed pregnancies. With the exception of day 135, all fetuses were collected by hysterotomy (gestation = 133 ± 2 days). Various weights and measurements were taken. Although fetal sizes varied widely, crown-rump, crown-heel, upper and lower arm and leg lengths, hand and foot lenghts, and fetal organ weights correlated well with fetal age. Key changes in gross brain morphology and skeletal ossification of cranium, pelvis, tarsals, carpals, and epiphyses were noted. Sternabrae ossification proved highly variable, while ossification in the otic capsule and associated ear structures correlated well with age. These data provide a normal base for studies using fetal developmental parameters whether the purpose is to determine gestational age or to design future studies.

The sit-up position for breast reconstruction.

For breast reconstruction, we have found that breast symmetry can be created optimally when the patient is brought to a totally upright position with the trunk vertical and perpendicular to the floor. The size of the opposite breast can better be appreciated. Nipple and areola position can be determined accurately even with the patient asleep if she is brought to the upright position. The safety of the upright positioning is documented in our series but depends on planning with the anesthesiologist preoperatively and allowing time to move the patient cautiously. An anesthesia technique for intraoperative vertical positioning is reported.

Severe respiratory depression in the obstetric patient after intrathecal meperidine or sufentanil.

Two cases of severe respiratory depression in the obstetric population are presented. The first occurred after intrathecal injection of a modest dose (50 mg) of meperidine. The second followed intrathecal administration of 10 microg of sufentanil after intravenous fentanyl. These cases illustrate the potential gravity of this complication and highlight the need for caution when giving intrathecal opioid following parenteral opioid administration.

Transient radicular irritation after hyperbaric lidocaine spinal anesthesia in parturients.

Two cases of transient radicular irritation in pregnant patients are presented. Both cases involve the combination of spinal anesthesia employing hyperbaric 5% lidocaine and a small gauge pencilpoint needle as well as the surgery being performed in the lithotomy position. We recommend that until the potential for lidocaine-induced neuroradicular irritation under these circumstances is evaluated prospectively, hyperbaric lidocaine should not be used for cases in which a small gauge spinal needle is employed and the patient is placed in the lithotomy position.

Fate of 2,2,2-trichloroacetaldehyde (chloral hydrate) produced during trichloroethylene oxidation by methanotrophs.

Four different methanotrophs expressing soluble methane monooxygenase produced 2,2,2-trichloroacetaldehyde, or chloral hydrate, a controlled substance, during the oxidation of trichloroethylene. Chloral hydrate concentrations decreased in these cultures between 1 h and 24 h of incubation. Chloral hydrate was shown to be biologically transformed to trichloroethanol and trichloroacetic acid by Methylosinus trichosporium OB3b. At elevated pH and temperature, chloral hydrate readily decomposed and chloroform and formic acid were detected as products.

Reproduction of the greater bushbaby (Galago crassicaudatus panganiensis) under laboratory conditions.

A breeding colony of Galago crassicaudatus panganiensis was maintained under laboratory conditions for four years, August 1971 to July 1975. The estrous cycle and the gestation period averaged 39 and 132 days in length, respectively. Twinning occurred in 14.5% of the pregnancies. No postpartum estrus was observed, although 1.2 conceptions per female per year were recorded. Breeding occurred throughout the year. Puberty occurred between ten and 18 months of age, the first conception occurred between 15 and 18 months of age, and reproductive capability decreased rapidly between ten and 12 years of age.

Purification and characterization of toluene 2-monooxygenase from Burkholderia cepacia G4.

Recent in vivo studies indicate that ring monooxygenation is a widespread mechanism by which bacteria metabolize aromatic hydrocarbons and obtain carbon and energy. In this study, toluene 2-monooxygenase from Burkholderia (formerly Pseudomonas) cepacia G4 was purified to homogeneity and found to be a three-component enzyme system. The reconstituted enzyme system oxidized toluene to o-cresol and o-cresol to 3-methylcatechol, an important intermediate for growth of the bacterium on toluene. Steady-state kinetic parameters measured for the water-soluble substrate o-cresol were a Km of 0.8 microM and a Vmax of 131 nmol min-1 (mg of hydroxylase protein)-1. The three protein components were (1) a 40 kDa polypeptide containing one FAD and a [2Fe2S] cluster, (2) a 10.4 kDa polypeptide that contained no identifiable metals or organic cofactors, and (3) a 211 kDa alpha 2 beta 2 gamma 2 component containing five to six iron atoms. The 40 kDa flavo-iron-sulfur protein oxidized NADH and transferred electrons to cytochrome c, dyes, and the alpha 2 beta 2 gamma 2 component. It is analogous to other NADH oxidoreductase components found in a wide range of bacterial mono- and dioxygenases. The 10.4 kDa component, added to the other two components and NADH, increased toluene oxidation rates 10-fold. The alpha 2 beta 2 gamma 2 component was indicated to contain the site for toluene binding and hydroxylation by the following observations: (1) tight binding to a toluene affinity column; (2) oxidation of toluene after reduction of the protein with dithionite and adding O2; (3) H2O2-dependent toluene oxidation and catalase activity; and (4) spectroscopic studies of the iron atoms in the component. The alpha 2 beta 2 gamma 2 component had no significant absorbance in the visible region. EPR spectroscopy yielded a signal at g = 16 upon addition of > 2 equiv of electrons per 2 Fe atoms. Taken with the quantitation of five to six iron atoms, the data suggest that the alpha 2 beta 2 gamma 2 component contains two binuclear iron centers. In total, the structural, spectroscopic, and catalytic features of toluene 2-monooxygenase are reminiscent of soluble methane monooxygenase obtained from methanotrophic bacteria. The two enzyme systems also differ in many subtle ways; for example, they oxidize toluene with completely different regiospecificity.

Trichloroethylene oxidation by purified toluene 2-monooxygenase: products, kinetics, and turnover-dependent inactivation.

Trichloroethylene is oxidized by several types of nonspecific bacterial oxygenases. Toluene 2-monooxygenase from Burkholderia cepacia G4 is implicated in trichloroethylene oxidation and is uniquely suggested to be resistant to turnover-dependent inactivation in vivo. In this work, the oxidation of trichloroethylene was studied with purified toluene 2-monooxygenase. All three purified toluene 2-monooxygenase protein components and NADH were required to reconstitute full trichloroethylene oxidation activity in vitro. The apparent Km and Vmax were 12 microM and 37 nmol per min per mg of hydroxylase component, respectively. Ten percent of the full activity was obtained when the small-molecular-weight enzyme component was omitted. The stable oxidation products, accounting for 84% of the trichloroethylene oxidized, were carbon monoxide, formic acid, glyoxylic acid, and covalently modified oxygenase proteins that constituted 12% of the reacted [14C]trichloroethylene. The stable oxidation products may all derive from the unstable intermediate trichloroethylene epoxide that was trapped by reaction with 4-(p-nitrobenzyl)pyridine. Chloral hydrate and dichloroacetic acid were not detected. This finding differs from that with soluble methane monooxygenase and cytochrome P-450 monooxygenase, which produce chloral hydrate. Trichloroethylene-dependent inactivation of toluene 2-monooxygenase activity was observed. All of the protein components were covalently modified during the oxidation of trichloroethylene. The addition of cysteine to reaction mixtures partially protected the enzyme system against inactivation, most notably protecting the NADH-oxidoreductase component. This suggested the participation of diffusible intermediates in the inactivation of the oxidoreductase.

Temporomandibular malformations in the bonnet monkey (Macaca radiata) fetus following maternal ingestion of thalidomide.

Structural abnormalities of the temporomandibular region were seen and described in the fetal bonnet monkey (Macaca radiata) after maternal ingestion of thalidomide. A single oral dose of 10 mg/kg of thalidomide was given on day 24, 25, 26, 27, 28 or 29 or 30 mg/kg on day 25 or 28 of gestation to 25 pregnant monkeys (day of mating = 0). The skeletons, processed and stained with alizarin red S, were examined for gross changes in configuration of craniofacial bones. Temporomandibular defects, in varying severity, were seen in four 70-day-old and seven 100-day-old fetuses associated with a single treatment between day 24 and day 28. The highest risk appeared to occur following treatment on day 25 (4/5) and the lowest on day 28 (2/8). Characteristic expression of the defects included 1) temporo-zygomatic hyperplasia involving the articular tubercle and postglenoid process and 2) mandibular hypoplasia, primarily of the condyles and the angular and coronoid processes. Micrognathia, apertognathia, and partial extraarticular ankylosis were seen. Although the sensitivity periods of the temporomandibular and ear regions seem to overlap and the embryonic origins are similar, the defects of each region appeared to be independent. Although the ear demonstrated a definite temporal relationship characterized by a decrease in severity in the expression of the ear malformations with respect to increased gestational age at the time of thalidomide insult, the temporomandibular region failed to exhibit a specific pattern. Although the temporo-zygomatic hyperplasia was dissimilar to any reported human condition, the mandibular changes were similar.