Comparison of recovery of sheep, goats, and calves from reversible electrical head-only and head-to-body stunning for halal meat production.

AIMS: To compare the recovery of lambs, goats, and calves from head-only (HO) or high-frequency head-to-body stunning and evaluate the complementary use of behaviour and electroencephalography (EEG) to assess return to consciousness after electrical stunning in these species. METHODS: Six-month-old lambs, adult goats and calves (< 7 days old) were subjected to reversible head-only stunning (50 Hz, 1 A, 2 seconds) or reversible high-frequency head-to-body stunning (RHTB: HO followed by 2,000 Hz, 2 A, 4-second stun to body). Following stunning, behavioural recovery was assessed in 21 lambs, 22 goats, and 20 calves. Latencies to first perform behaviours (end of convulsions, head lift, attempt to right, successful righting, attempt to stand, successful standing) after stunning were scored from video recordings. Recovery of electrical brain activity indicative of consciousness was assessed using EEG in a separate cohort of minimally-anaesthetised lambs, goats and calves (n = 20 per species). EEG traces collected before and after stunning were classified as normal, epileptiform, isoelectric, or transitional activity. Following stunning, the duration of epileptiform and isoelectric activity combined (states of brain activity incompatible with conscious awareness) was calculated, as was latency to return of normal (pre-stun) EEG. RESULTS: The RHTB stun was reversible in all three species, although one sheep failed to recover and was euthanised. Both methods caused tonic and clonic convulsions in all species. Behavioural recovery of sheep and calves was similar for both methods while goats took longer to recover from RHTB than HO stunning. There was no evidence of differences between methods in the duration of EEG incompatible with consciousness or the latency to recovery of normal EEG. CONCLUSIONS: Head-to-body stunning as applied here produced a reversible electrical stun in lambs, adult goats and young calves, although the benefits in terms of meat quality and operator safety are uncertain. Goats took longer to recover behaviourally from head-to-body stunning, possibly due to disrupted motor function, but there was no indication that post-stun unconsciousness lasted longer than following head-only stunning in any species. The normal behaviour for the animals' developmental age should be considered when deciding on behavioural indicators of recovery. The minimal anaesthesia model provided excellent quality EEG data that was valuable for interpretation of the behavioural responses. CLINICAL RELEVANCE: For the purposes of pre-slaughter stunning of sheep, goats and young calves, recovery appears comparable between the two methods, with all but 1/63 animals in the behaviour study recovering normal function.

Indicators of dehydration in healthy 4- to 5-day-old dairy calves deprived of feed and water for 24 hours.

Our objective was to identify practical indicators of calf dehydration that could be used in an industry context. Eleven healthy 4-d-old commercial dairy calves were fed 2 L of mixed colostrum, then deprived of food and water for 24 h. Total body water was determined in the fed state using the deuterium dilution method. Body weight, along with a range of behavioral and physiological variables, was recorded 1 h after feeding, then at 90-min intervals through to 24 h. Blood samples were collected at every second sampling to assess changes in plasma hemoglobin, hematocrit, and osmolality. Linear mixed-effects models were used to explore associations between hydration status (% body water) and outcome variables. All calves remained bright and alert with good suckling reflexes throughout the 24-h period. After 24 h, total body water had decreased by an average of 8.4% (standard error 1.18), consistent with mild to moderate dehydration. Skin tent return time, capillary refill time, and detectable enophthalmos were associated with hydration status. Calves with skin tent return times of 3 s or longer were 4.4 percentage points less hydrated than those with return times of less than 3 s. Similarly, a capillary refill time of 3 s or longer was associated with a 4.3 percentage point reduction in hydration compared with refill times of less than 3 s. Calves with detectable enophthalmos (≥1 mm) were 3.5 percentage points less hydrated than those without enophthalmos. The skin tent, capillary refill, and enophthalmos tests are all relatively simple to perform and, although requiring the calf to be briefly restrained, can easily be performed by a single operator. The outcome of these tests was relatively consistent, in that calves above the threshold in any test were 3.5 to 4.5% less hydrated than calves below the threshold. As such, these tests may be of practical utility to identify calves with mild to moderate dehydration in an industry setting.

Effects of Obesity on the Regulation of Macrophage Population in the Prostate Tumor Microenvironment.

Obesity is associated with a greater risk of prostate cancer mortality. However, the mechanisms connecting obesity to the progression of prostate cancer remain unknown. This study determined the impact of obesity on macrophage recruitment and tumor-associated macrophage (TAM) polarization in the prostate tumor microenvironment, since a high concentration of TAMs in tumors has been linked to progression in prostate cancer. We utilized an in vitro model in which pre-adipocytes, prostate cancer cells, and macrophages were exposed to sera from obese or nonobese men, or conditioned media generated under obese or nonobese conditions. Matrigel invasion chambers were used to assess macrophage recruitment in vitro, and immunohistochemical analysis evaluated recruitment in a PTEN knockout mouse model. qPCR was used to measure mRNA levels of CCL2, COX-2, IL-10, TGF-beta, VEGF-A, arginase-1, and MMP-9. PGE2 production was measured by ELISA. Obesity increased macrophage and TAM recruitment, and increased mRNA levels of TAM markers in macrophages. Similarly, obese conditions increased CCL2 and COX-2 expression, as well as PGE2 levels in prostate cancer cells. COX-2 inhibition resulted in lower expression of obesity-induced TAM markers. Our data suggest that obesity promotes macrophage infiltration into the prostate tumor microenvironment, and induces TAM polarization through the COX-2/PGE2 pathway.

[Effect of ethanol on synthesis of serine and exchange of methyl groups in hepatocytes by NMR spectroscopy].

The method of NMR spectroscopy was used to investigate the role of voltage-dependent anion channels in the outer mitochondrial membrane in the mechanism of ethanol hepatotoxicity using the synthesis of serine and exchange of methyl groups in hepatocytes metabolizing 13C-labeled glycine. Here we present and describe a methodological approach developed for the independent monitoring of the synthesis of serine in two intracellular compartments: the cytoplasm and mitochondria of intact hepatocytes, and quantification of different serine isotopomers synthesized in hepatocytes from 13C-labeled glycine. The data obtained indicate that the treatment of cells with ethanol as well as cysteamine (specific inhibitor of mitochondrial synthesis of serine) suppressed the level of mitochondria but not cytoplasmic serine isotopomers. It is concluded that the decrease in the production of mitochondrial serine isotopomers in hepatocytes exposed to ethanol can be caused not only by decreased permeability of the outer mitochondrial membrane due to the closure of voltage-dependent anion channels and suppression of the exchange of substrates of serine synthesis in mitochondria but also by the restoration of the cytoplasmic and/or mitochondrial pool of pyridine nucleotides (NADH) during the oxidation of ethanol. Our work reveals a new mechanism of action of ethanol (alcohol intoxication) in hepatocytes through the regulation of glycine metabolism and opens new possibilities in the treatment of alcohol poisoning.

[The role of the voltage-dependent anion channels in the outer membrane of mitochondria in the regulation of cellular metabolism].

The role of the voltage-dependent anion channels (VDAC) harbored in the outer membrane of mitochondria in the regulation of cellular metabolism was investigated using an experimental model of ethanol toxicity in cultured hepatocytes. It was demonstrated that ethanol inhibits State 3 and uncoupled mitochondrial respirations, decreases the accessibility of mitochondrial adenylate kinase localized in the intermembrane space of mitochondria, and suppresses ureagenic respiration and synthesis of urea in cultured hepatocytes. Increasing the permeability of the outer mitochondrial membrane with closed VDAC with high concentrations of digitonin (> 80 microM), which creates pores in the membrane, allowing the alternative bypass of closed VDAC, and restores all reactions suppressed with ethanol. It is concluded that the effect of ethanol in hepatocytes leads to global loss of mitochondrial functions due to the closure of VDAC, which limits the free diffusion of metabolites into the intermembrane space of mitochondria. Our studies demonstrated that ethanol affects the main mitochondrial functions and revealed the role of VDAC channels in the outer mitochondrial membrane in the regulation of liver specific intracellular processes such as ureagenesis. The data obtained can be used for the development of pharmaceutical drugs that prevent the closure of VDAC in mitochondria of ethanol oxidizing liver, thus protecting liver tissue from the hepatotoxic action of alcohol.

Influence of human development and predators on nest survival of tundra birds, Arctic Coastal Plain, Alaska.

Nest predation may influence population dynamics of birds on the Arctic Coastal Plain (ACP) of Alaska, USA. Anthropogenic development on the ACP is increasing, which may attract nest predators by providing artificial sources of food, perches, den sites, and nest sites. Enhanced populations or concentrations of human-subsidized predators may reduce nest survival for tundra-nesting birds. In this study, we tested the hypothesis that nest survival decreases in proximity to human infrastructure. We monitored 1257 nests of 13 shorebird species and 619 nests of four passerine species at seven sites on the ACP from 2002 to 2005. Study sites were chosen to represent a range of distances to infrastructure from 100 m to 80 km. We used Cox proportional hazards regression models to evaluate the effects of background (i.e., natural) factors and infrastructure on nest survival. We documented high spatial and temporal variability in nest survival, and site and year were both included in the best background model. We did not detect an effect of human infrastructure on nest survival for shorebirds as a group. In contrast, we found evidence that risk of predation for passerine nests increased within 5 km of infrastructure. This finding provides quantitative evidence of a relationship between infrastructure and nest survival for breeding passerines on the ACP. A posteriori finer-scale analyses (within oil field sites and individual species) suggested that Red and Red-necked Phalaropes combined (Phalaropus fulicarius, P. lobatus) had lower productivity closer to infrastructure and in areas with higher abundance of subsidized predators. However, we did not detect such a relationship between infrastructure and nest survival for Semipalmated and Pectoral Sandpipers (Calidris pusilla, C. melanotos), the two most abundant shorebirds. High variability in environmental conditions, nest survival, and predator numbers between sites and years may have contributed to these inconsistent results. We recommend targeted management actions to minimize anthropogenic effects and suggest new research needed on this issue as expanding development is planned for the ACP of Alaska. In particular, we recommend research on demography of key predators and their importance with respect to nest survival, and experimental studies that better address challenges posed by high natural variability.

Electroencephalographic responses to tail clamping in anaesthetized rat pups.

We investigated electroencephalographic (EEG) responses to tail clamping in lightly anaesthetized rat pups (5-22 days) in order to determine the ontogeny of EEG activity and at what age they may be capable of experiencing pain. Median frequency (F50) and spectral edge frequency (F95) of the power spectrum in the range of 1-30 Hz were determined before and after the application of a noxious stimulus and power spectra were compared by multivariate analysis. There was a postnatal increase in EEG power as, before clamping, pups aged 5-7 days exhibited isoelectric traces, whereas those aged 12-14 days and 21-22 days had intermittent EEG activity where the power in all frequencies was significantly lower at the former than at the latter age. Pups aged 5-7 days exhibited no EEG response to clamping in view of their isoelectric traces. Pups aged 12-14 days showed a significant decrease in F95 (P=0.002), whereas those aged 21-22 days showed highly significant reduction in F50 and F95 (P=0.028 and P<0.001, respectively) as well as changes in EEG power of specific frequencies after clamping. The results and related literature suggest that rat pups aged 5-7 days and younger are not likely to perceive pain and that the ability to perceive pain develops gradually between postnatal ages 12-14 days and 21-22 days.

Synergistic depression of NMDA receptor-mediated transmission by ketamine, ketoprofen and L-NAME combinations in neonatal rat spinal cords in vitro.

BACKGROUND AND PURPOSE: Spinal N-methyl-D-aspartate (NMDA) receptor/cyclooxygenase (COX) and nitric oxide synthase (NOS) pathways play a major role in nociceptive processing, and influencing them simultaneously may induce synergistic analgesia. This study determined the spinal antinociceptive interactions between ketamine (NMDA receptor channel blocker), ketoprofen (COX inhibitor) and L-NAME (NOS inhibitor) combinations. EXPERIMENTAL APPROACH: Using an in vitro neonatal rat spinal cord preparation, two A-fibre-mediated reflexes, the monosynaptic reflex (MSR) and the low-intensity excitatory postsynaptic potential (epsp), and one C-fibre-mediated reflex, the high-intensity epsp, were evoked electrically. The effect of drugs and drug combinations on these reflexes was assessed and the type of interaction determined by isobolographic analysis. KEY RESULTS: Infusion of ketamine alone decreased all three reflexes. That of ketoprofen decreased both the low and the high-intensity epsp only. Infusion of L-NAME alone produced no significant effects. Co-infusion of fixed ratios of IC(40) fractions of both (ketamine+ketoprofen) and (ketamine+L-NAME) were synergistic for depressing the low and the high-intensity epsps. The interaction was sub-additive for both combinations on the MSR. The only significant effect for the (ketoprofen+L-NAME) combination was synergism on the high-intensity epsp. CONCLUSIONS AND IMPLICATIONS: All three combinations synergistically depressed nociceptive spinal transmission, and both ketamine and ketoprofen and ketamine and L-NAME combinations did so with potentially decreased motor side effects. If such combination profiles also occur in vivo, the present findings raise the possibility of ultimate therapeutic exploitation of increased analgesia with fewer side effects.

Comparative effects of halothane, isoflurane, sevoflurane and desflurane on the electroencephalogram of the rat.

Inhalant anaesthetic agents are commonly used in studies investigating the electroencephalographic (EEG) effects of noxious stimuli in animals. Halothane causes less EEG depression than isoflurane, however, the EEG effects of halothane, isoflurane, sevoflurane and desflurane have not been compared in the same model. This study aimed to compare the EEG effects of these inhalational agents in the rat. Forty male Sprague-Dawley rats were assigned to four groups and anaesthetized with halothane, isoflurane, sevoflurane or desflurane. EEG was recorded from the left and right somatosensory cortices for 5 min at three different multiples of minimal alveolar concentration (MAC) (1.25, 1.5 and 1.75). Median, 95% spectral edge frequency and total power were derived and a single mean value for each was calculated for the first 60 s of each recording period. When the raw EEG contained burst suppression (BS), the BS ratio (BSR) over 60 s was calculated. No BS was found in EEG recorded from the halothane group at any concentration. BS was present at all concentrations with the other anaesthetic agents. BS was almost complete at all concentrations of isoflurane, whereas BSR increased significantly with increasing concentrations of sevoflurane and desflurane. No significant differences were found between the BSR due to the 1.75 MAC multiple of isoflurane, sevoflurane or desflurane. Halothane causes significantly less depression of cortical activity than the newer inhalant agents at equivalent multiples of MAC. These data support the hypothesis that halothane has a fundamentally different mechanism of action than the other inhalant agents.

Export of dissolved organic carbon from a ponded freshwater marsh receiving diverted Mississippi River water.

A series of diversion projects has been implemented to reintroduce Mississippi River water into Louisiana's coastal wetlands in order to reduce wetland loss. The export of dissolved organic carbon (DOC) was measured in a 3,700-ha ponded freshwater marsh that receives diverted Mississippi River water. Results show that highly organic marsh soil and plant material are a source of DOC. DOC, on average, was 3 mg/l greater in outlet water as compared to the concentration in river water entering the wetland. DOC in water leaving the marsh was higher in summer months, with a concentration up to 18 mg/l. Based on a discharge of 1,000 ft3/sec (28.3 m3/sec), it was estimated that the equivalent of 7,335 kg/day of DOC would be exported from the marsh into Lake Cataouatche, located in the northern portion of the Louisiana Barataria Basin estuary. Results suggest that river diversion would likely increase the export of DOC from the marsh as compared to normal transport associated with rainfall and tidal exchange.

Comparison of neural histomorphology in tail tips from pigs docked using clippers or cautery iron.

Tail docking of pigs is commonly performed to reduce the incidence of unwanted tail-biting behaviour. Two docking methods are commonly used: blunt trauma cutting (i.e. using side clippers), or cutting and concurrent cauterisation using a hot cautery iron. A potential consequence of tail amputation is the development of neuromas at the docking site. Neuromas have been linked to neuropathic pain, which can influence the longer-term welfare of affected individuals. To determine whether method of tail docking influences the extent of neuroma formation, 75 pigs were allocated to one of three treatments at birth: tail docked using clippers; tail docked using cautery iron; tail left intact. Tail docking was performed at 2 days of age and pigs were kept under conventional conditions until slaughter at 21 weeks of age. Tails were removed following slaughter and subjected to histological examination. Nerve histomorphology was scored according to the following scale: 1=discrete well-organised nerve bundles; 2=moderate neural proliferation and disorganisation affecting more than half of the circumference of the tail; 3=marked neural proliferation to form almost continuous disorganised bundles or non-continuous enlarged bundles compressing the surrounding connective tissue. Scores of 2 or 3 indicated neuroma formation. Scores were higher in docked pigs than undocked pigs (P<0.001), but did not differ between pigs docked using clippers and those docked using cautery (P=0.23). The results indicate that tail docking using either clippers or cautery results in neuroma formation, thus having the potential to affect long-term pig welfare.

Depression of NMDA-receptor-mediated segmental transmission by ketamine and ketoprofen, but not L-NAME, on the in vitro neonatal rat spinal cord preparation.

Activation of spinal N-methyl-D-aspartate (NMDA) receptors and then the nitric oxide and the arachidonic acid pathways is important in pain transmission. This study assessed the effects of the NMDA receptor channel blocker ketamine, the nitric oxide synthase inhibitor L-NAME, and the cyclooxygenase inhibitor ketoprofen in nociceptive transmission using an in vitro neonatal rat spinal cord preparation. Supramaximal electrical stimulation of the dorsal root evoked the A-fibre- and C-fibre-mediated high intensity excitatory postsynaptic potential (EPSP) in the ipsilateral ventral root. Low intensity stimulation evoked the A-fibre-mediated monosynaptic compound action potential (MSR) superimposed on the low intensity EPSP. Both the low intensity EPSP and the high intensity EPSP contain NMDA-receptor-mediated components. Only ketamine and ketoprofen depressed the synaptic responses. Ketamine depressed all three spinal reflexes with IC(50) values (with 95% CI) of 10.80 microM (5.97 to 19.54 microM) for the MSR, 8.29 microM (4.53 to 14.17 microM) for the low intensity EPSP, and 5.35 microM (3.05 to 9.40 microM) for the high intensity EPSP. Ketoprofen depressed the low intensity EPSP and the high intensity EPSP only; IC(50) values (with 95% CI) were 354.5 microM (217.5 to 576.8 microM) and 302.7 microM (174.0 to 526.7 microM), respectively. Reflexes recovered after drug washout. These data demonstrated that ketamine and ketoprofen, but not L-NAME, depressed NMDA-mediated nociceptive transmission in spinal cord preparations from neonatal rats.

A scientific comment on the welfare of domesticated ruminants slaughtered without stunning.

This review provides a scientific comment on the welfare of ruminants slaughtered by ventral-neck incision without stunning. Evidence is derived from studies of calves, sheep and goats. Reference is also made to findings in other mammals including humans. Pain is an inherently subjective experience and only indirect indices are available in animals. Neurophysiological tools are widely used in humans to assess pain and have demonstrated that electroencephalographic (EEG) variables correlate well with subjective evaluations of pain. These neurophysiological tools have also been applied in animal studies. In humans pain is associated with major cutting injuries and it is widely accepted that farm animals also experience pain due to such injuries. Overwhelming international scientific opinion has long been that slaughter by neck incision of conscious animals causes pain. A series of studies in calves demonstrated that slaughter by ventral-neck incision is likely to be perceived as painful. It is proposed that, as in cattle, non-stunned sheep and goats would experience pain in a similar manner. The precise assessment of the point after slaughter at which non-stunned animals become insensible remains a major methodological challenge. In sheep it is at least 2-8 seconds, but may be 8-20 seconds in duration. In cattle the mean duration is similar, but can commonly be extended to longer than 60 seconds with occasional instances of even greater durations. Taken together, these findings indicate that because the slaughter of cattle, sheep and goats by ventral-neck incision without prior stunning is likely to cause pain, this poses a risk to animal welfare.

Spontaneous electroencephalographic changes in a castration model as an indicator of nociception: a comparison between donkeys and ponies.

REASONS FOR PERFORMING STUDY: Donkeys are believed to be less demonstrative of pain than ponies. Research into comparative sensory processing between these species is required to elucidate these behavioural differences. OBJECTIVES: To compare changes in the electroencephalogram (EEG) recorded during castration between donkeys and ponies. STUDY DESIGN: Prospective clinical study. METHODS: Six ponies and 6 donkeys were castrated under halothane anaesthesia after acepromazine premedication and thiopental anaesthetic induction. Markers were inserted into the EEG recording at the time of skin incision (skin) and emasculation (emasc) for both testicles (T1 and T2) during a closed castration. Raw EEG data were analysed and the EEG variables median frequency (F50 ), total power (Ptot ) and spectral edge frequency (F95 ) derived using standard techniques. Baseline values of F50 , Ptot and F95 for each animal were used to calculate the percentage change from baseline at T1skin, T2skin, T1emasc and T2emasc. RESULTS: Decreased F50 values relative to baseline were observed in 4 ponies and 2 donkeys across all castration time points. In the remaining animals, the F50 value increased compared with baseline. Both donkey and pony groups showed an overall decrease in Ptot values compared with baseline at T1skin, but the magnitude of the decrease was significantly less (P = 0.004) in ponies than in donkeys. Donkeys demonstrated an overall greater increase (P = 0.05) in F95 values at T1skin relative to baseline compared with ponies. CONCLUSIONS: Electroencephalographic responses to the noxious stimulus of castration were noted in both donkeys and ponies. Donkeys demonstrated a greater change in Ptot in response to castration than ponies; thus, donkeys appear to demonstrate a cerebral cortical response to a noxious stimulus that is similar to or greater than that in ponies, suggesting that their subtle behavioural expression of pain is not due to a difference in cortical processing of noxious sensory stimuli.

Proton magnetic resonance spectroscopy and outcome in term neonates with chorioamnionitis.

OBJECTIVE: Evaluate brain metabolites, which reflect neuroinflammation, and relate to neurodevelopmental outcomes in healthy term neonates exposed to chorioamnionitis. STUDY DESIGN: Thirty-one healthy term neonates with documented fetal inflammatory response after maternal chorioamnionitis underwent magnetic resonance spectroscopy (MRS), with voxels placed in basal ganglia (BG) and frontal white matter. Bayley III examinations were performed at 12 months of age. RESULT: Infants with below average outcomes did not show the same increase in NAA/Cho ratios postnatally as the group with normal outcomes. Decreased NAA/Cho and increased Lac/Cr in BG correlated with lower motor and cognitive composite scores, respectively, controlling for postnatal age. In males, increased lactate/NAA in BG were associated with lower motor scores. Funisitis severity was associated with decreased NAA/Cho and increased mI/NAA in males. CONCLUSION: In healthy term newborns with chorioamnionitis, MRS ratios shortly after birth may provide evidence of occult neuroinflammation, which may be associated with worse performance on 1-year neurodevelopmental tests.

Job analysis 1992: infection control practitioner.

The Certification Board of Infection Control directed its Research Subcommittee to compose a Job Analysis Committee in 1991. This 9-member Job Analysis Committee, in collaboration with Applied Measurement Professionals, Inc., conducted a job analysis of ICPs during 1992. The reassessment of the previous Certification Board of Infection Control task analysis, formation of a job-analysis survey tool, and the actual job-analysis process and its results are described in this article. The previous and newly revised test specification outlines are compared. The national Certification Examination for Infection Control for November 1993 will reflect the efforts of this endeavor.

Multidrug resistance-1 gene expression does not increase during tumor progression in the MGH-OGS murine osteosarcoma tumor model.

In addition to its possible role in drug resistance, expression of the multidrug resistance-1 gene may also be associated with a more malignant phenotype and tumor progression. This study evaluated its expression during tumor progression in the MGH-OGS transplantable murine osteosarcoma tumor model. Three variables of tumor progression were analyzed: tumor size, local recurrence, and metastasis. With a highly sensitive reverse transcription-polymerase chain reaction method, mRNA levels of multidrug resistance-1 were compared in primary tumors of different sizes. In addition, the levels were compared in primary, locally recurrent, and metastatic tumors isolated from individual mice. No significant difference was found in the levels of expression with increasing primary tumor size. In addition, the levels in primary, locally recurrent, and metastatic tumors were not significantly different. Our results indicate that--at least in the MGH-OGS tumor model, which is analogous to the majority of spontaneously occurring human osteosarcomas in that it has low levels of multidrug resistance-1/P-glycoprotein and is sensitive to doxorubicin--there is no evidence of upregulation of multidrug resistance-1 expression during tumor progression.

Pulmonary toxicity of 2-methylnaphthalene: lack of a relationship between toxicity, dihydrodiol formation and irreversible binding to cellular macromolecules in DBA/2J mice.

Intraperitoneal doses of 2-methylnaphthalene (2-MN) have been shown to cause pulmonary toxicity in DBA/2J mice. Pretreatment with the monooxygenase inducers sodium phenobarbital and 3-methylcholanthrene (3-MC) failed to protect the DBA/2J mice from the toxic effect of 2-methylnaphthalene. Pretreatment of DBA/2J mice with the monooxygenase inhibitors, SKF 525-A and piperonyl butoxide also failed to enhance or attentuate the pulmonary lesions. Pulmonary and hepatic microsomes from DBA/2J mice metabolized 2-methylnaphthalene to three dihydrodiols, 2-naphthyl alcohol and other unidentified metabolites. Kidney microsomes produced 2-naphthyl alcohol but no detectable dihydrodiols. In comparison to control animals, hepatic microsomes from animals pretreated with sodium phenobarbital produced more of the least polar dihydrodiol, while amounts of the other two dihydrodiols were unaffected. 3-Methylcholanthrene, piperonyl butoxide and diethylmaleate failed to affect dihydrodiol formation in both pulmonary and hepatic microsomes. After the administration of a lung toxic dose (400 mg/kg, i.p.) of 2-MN, irreversible binding was highest in the liver, followed by the kidney, the lung and lastly skeletal muscle. Of the pretreatments given to the mice, only phenobarbital demonstrated a significant effect, and this elevation was apparent only in the liver. A pulmonary toxic dose of 2-MN (400 mg/kg, i.p.) administered to DBA/2J mice significantly depleted reduced GSH in the liver and lung and to a lesser extent, in the kidney. There appeared no good correlation between the pulmonary toxicity of 2-MN-dihydrodiol and/or alcohol formation or the in vivo irreversible binding to macromolecules. These results are compared with those reported previously in C57BL/6J mice.

Isolation of cholesterol oxidation products from animal fat using aminopropyl solid-phase extraction.

Cholesterol oxidation products were separated from triglycerides and cholesterol in a single step on an aminopropyl solid-phase extraction column. The products were purified by subsequent transesterification and saponification, derivatized to trimethylsilyl ethers and analyzed by gas chromatography. Heated cholesterol-containing fat samples were autoxidized by bubbling air through them. When the flow-rate of air was set at 100 ml/min, the concentration of cholesterol oxidation products in the fat increased to a maximum after 1-2 h and then decreased to almost a zero level after 8 h. The concentration of cholesterol oxidation products in the fat increased over a similar time period, without reaching a maximum, when the flow-rate of air was decreased to 5 ml/min.