A. M. Symons

Pineal function in the sheep: evidence for a possible mechanism mediating seasonal reproductive activity.

The 24-h profiles of plasma melatonin in the intact ewe in natural light indicate that a bimodal pattern of secretion is frequently present in oestrus, whereas a single dark-phase peak is characteristic of anoestrus. Based on these findings, a mechanism for the possible pineal-mediated control of seasonal breeding is proposed.

The effects of alcohol on weight gain and the hypothalamic-pituitary-gonadotrophin axis in the maturing male rat.

Abstract Male rats allowed food ad lib. and 15% (v/v) alcohol in place of water ad lib. from 3 to 10 weeks of age were found to have a slightly slower growth rate than control animals. This was due to a lower caloric intake. The normal increase in basal luteinizing hormone (LH) levels that takes place during maturation in the male rat was completely abolished by alcohol feeding. That this was not only (or merely) due to impaired weight gain was suggested by the observation that intubation of alcohol (0.28 g) into 150-g male rats established that alcohol was capable of lowering circulating LH levels within 1 hr. The sensitivity of the anterior pituitary to 100 ng exogenous luteinizing hormone-releasing hormone (LH-RH) was the same in alcohol-fed and control rats, suggesting that the alcohol effect on the reproductive endocrine system is at the hypothalamic or higher neural centres.

Effects of ether anaesthesia and fasting on various cytochromes P450 of rat liver and kidney

Fed and fasted, male, Wistar albino rats exposed to light ether anaesthesia and killed immediately or after 30 or 120 min recovery were compared with non-anaesthetized rats for changes in liver and kidney cytochrome P450 (CYP) activities. In fed rats, liver total CYP (nmol/mg protein) decreased by 30% immediately after ether, but was restored to normal levels after 30 min recovery; in fasted rats, liver total CYP increased by 20% by fasting alone, then decreased by 65% immediately after ether, and recovered to only 70% of control at 2 hr after ether. Rat liver cytochrome P4501A (CYP1A; 7-ethoxyresorufin O-deethylase or EROD activity) and cytochrome P4502B (CYP2B; 7-pentoxyresorufin O-dealkylase or PROD activity) were decreased after ether anaesthesia, similar to those for total CYP. In contrast, rat liver cytochrome P4502E1 (CYP2E1), determined by p-nitrophenol hydroxylation, increased by 40% by ether anaesthesia alone, 70% by fasting alone and 140% by ether plus fasting; these increases were confirmed by the CYP2E1-mediated activation of nitrosopyrrolidine and by immunoblot analysis using antibody to CYP2E1. In rat kidney, losses of total CYP, CYP1A and CYP2B, and increases of CYP2E1, induced by ether anaesthesia, were much more marked in fasted (90% loss in total CYP, 30% increase in CYP2E1) than in fed rats (slight loss in total cytochrome P450, 30% increase in CYP2E1). As maximum losses of total CYP in liver of fasted rats exposed to ether occurred at the time of maximum increase of CYP2E1 and maximum rate of generation of reactive oxygen species (ROS), it is suggested that the increase of CYP2E1, resulting from its stabilization by fasting and ether, leads to generation of ROS, increase in lipid peroxidation and consequent loss of total CYP, associated with the hepatic and renal necrosis seen in ether intoxication and surgical trauma.

ROLE OF TISSUE GLUTATHIONE IN PREVENTION OF SURGICAL TRAUMA

1. Surgical trauma has been associated with pre-anaesthesia fasting, anaesthetic toxicity, haemorrhage, hypovolaemic shock, and other pathological phenomena. Tissue glutathione (GSH), thiobarbituric acid-reacting substances (TBAR), and radical-trapping activity (RTA) have been determined at various time intervals after fasting, anaesthesia, and also after hepatic ischaemia and reperfusion as a model for haemorrhage and hypovolaemic shock. 2. Light ether anaesthesia of rats resulted in an immediate (5 min) and progressive decrease in liver and kidney total glutathione (GSH and GSSG), which was much greater in animals that had been fasted for 20 h. TBARs, a measure of lipid peroxidation, in rat liver and kidney increased as total GSH decreased. Fasting (20 h) alone decreased tissue GSH by 50%, and increased TBAR 100%; fasting plus 30 min of ether anaesthesia decreased tissue glutathione by 80 to 85%, and increased TBAR by some 600%. 3. Liver ischaemia alone decreased total liver GSH by 20% in the fed rat, and 50% in the fasted rat. Ischaemia, followed by reperfusion, decreased liver total GSH by 70% in the fed rat, and 90% in the fasted rat. The ratio of GSH/GSSG decreased from 16 in control animals to 7 in the fasted ischaemic rat, then to 1 in the fasted, ischaemic rat reperfused for 90 min. RTA of liver closely paralleled liver total GSH levels. TBAR was increased by ischaemia alone (50-100%), but more (400%) by 90 min reperfusion. 4. A complex series of molecular mechanisms including: (1) GSH depletion; (2) induction of CYP2E1 activity; (3) generation of reactive oxygen species; (4) lipid peroxidation; (5) cytokine release; and (6) leucocyte activation, are advanced to account for the toxic phenomena of surgical trauma and multiple system organ failure.

Hepatic microsomal drug metabolism in the pregnant rat

1. Hepatic microsomal drug metabolism determined with the substrates aniline (p-hydroxylation), ethylmorphine (N-demethylation) and p-nitrobenzoic acid (reduction), decreased during gestation in the rat to 53-73% of non-pregnant control levels by day 20 of gestation. 2. Enzyme activity remained low at one day post-partum, but had returned to control non-pregnant levels by five days post-partum. 3. The total capacity of the liver to metabolize drugs remained unchanged or increased because liver weight was increased by up to 40% during pregnancy. 4. Changes in drug metabolism were not related to alterations in the concentration, substrate-induced binding affinity (Ks) or maximal spectral change (delta Amax) of cytochrome P-450. 5. Alterations in hepatic drug metabolism are possibly mediated via changes in microsomal phospholipids and/or the cytochrome P-450 spin-state equilibrium as as pregnancy was associated with a decrease in (a) microsomal total phospholipids, (b) the phosphatidylcholine to phosphatidylethanolamine ratio and (c) the high-spin form of ferricytochrome P-450.

Short-Term Variations of Circulating Melatonin in the Ewe

Melantonin levels have been studied in venous blood sampled at different frequencies (0.5‐, 2‐, 60‐min intervals) from intact ewes. All samples were taken during the dark phase of either natural or artificial photoperiods. In one experiment samples were taken simultaneously from both jugular veins to investigate the possible effects of “streaming” on the levels measured. Plasma cortisol was measured to ascertain whether or not the frequent removal of blood activated the ACTH stress axis. Plasma melantonin levels showed considerable variation with peaks of up to 365 pg/ml on a baseline of between 30 60 pg/ml. There was consistent evidence of intermittent peaks, the frequency of which increased with an increase in sampling frequency. Plasma cortisol showed no correlation with either the frequency or the amplitude of the melatonin peaks. When plasma samples were taken from both jugular veins a similar melatonin pattern was seen in the samples from both sides, but samples taken from the left jugular vein invariably showed higher levels than those taken from the right vein. This may be due to differential vascular drainage of the pineal to the two sides.

Free radical production at the site of an acute inflammatory reaction as measured by chemiluminescence

A foot-pad oedema model was used to investigate the presence of free radicals using a chemiluminescence method. This model is an example of a cell mediated hypersensitivity reaction. Male rats were inoculated in the scruff with Freunds Complete Adjuvant (FCA) on Day 0 and then challenged 6 days later with FCA in one hind paw. An acute inflammatory reaction was initiated over the following 96 hours and within 4 hours of induction, reactive oxygen species were detected in the inflamed tissue. A peak of chemiluminescence activity was seen 8 hours after the induction of the inflammatory reaction, well before maximum oedema was observed. Using mannitol, catalase and DABCO to elucidate the nature of the reactive oxygen species it was found that hydroxyl radicals, hydrogen peroxide and singlet oxygen all contributed to this burst of oxidative activity and are therefore probably involved with the process of lipid peroxidation and the severity of an inflammatory reaction.

Effects of neomycin on the biliary excretion and enterohepatic circulation of mestranol and 17β-oestradiol

The continued circulation of free steroids depends on their resorption from the gut following the hydrolysis of biliary conjugates. In this study, the bile duct of female Wistar albino rats was cannulated. Animals receiving labeled steroids or labeled bile intraductally also had the duodenum fitted with a cannula connected with a dosing syringe. In neomycin-treated rats, recirculation was impaired up to 50%. The deconjugation of mestranol and estradiol biliary conjugates was shown in vitro uponiincubation with rat caecal microorganisms, and the inhibition of such hydrolysis by neomycin was observed in vitro. Neomycin pretreatment reduced the biliary excretion of mestranol and estradiol after intraductal administration. It was thought that suppression of the gut microflora by neomycin was a major factor in the impairment of the intrahepatic circulation of mestranol and estradiol metabolites. This effect may be important regarding the half-life of estrogenic compounds of the contraceptive pill.

Inflammation, reactive oxygen species and cytochrome P450

Inflammation may ultimately result from damage to membrane lipids by reactive oxygen species (ROS) such as peroxide, superoxide anion, hydroxyl radical and singlet oxygen. This study compares some of the methods used to determine ROS—ethane exhalation, malondialdehyde quantified as thiobarbituric acid-reacting materials, and luminol-activated chemiluminescence (LAC)—and explores possible relationships with oedema formation in the rat foot-pad model. Iron nitrilotriacetate was the most effective of the model compounds tested in producing lipid peroxidation and ethane exhalation in mice. In the mouse and the rat, iron nitrilotriacetate caused increased ethane exhalation and concomitant increases in liver and kidney malondialdehyde. In the rat foot-pad oedema model, the challenge with Freunds complete adjuvant produced maximum malondialdehyde and maximum LAC in the inflamed paw 8 h after dosing, at which time oedema had also reached a high level. These effects were attributed mainly to hydroxyl radical and singlet oxygen. The inhibition of oedema by four anti-inflammatory drugs correlated well with LAC but less well with inhibition of malondialdehyde production. This study shows good agreement between different methods of determining ROS formation, and that inhibition of ROS formation in vivo is paralleled by a decrease in inflammation.

Autoxidative injury with loss of cytochrome P-450 following acute exposure of rats to fasting and ether anaesthesia

1. Exposure of fasted rats (20 h) to ether anaesthesia for 4 min resulted in increased exhalation of alkanes, an indication of lipid peroxidation in vivo. 2. Liver and kidney of the fasted rats anaesthetized with ether showed immediate 4-fold increases in luminol-amplified chemiluminescence, reaching maxima 30 min later, indicating the production of reactive oxygen species. 3. Liver and kidney cytosols of the fasted anaesthetized rats similarly showed immediate 4-fold increases of thiobarbituric acid-reactive material (malondialdehyde and other lipid peroxidation breakdown products) which attained maxima 60 min later. 4. Total cytochromes P-450 of liver and kidney of rats were decreased to 25-30% of control values after 20 h fasting and 4 min of ether anaesthesia, but were restored to normal levels 2 h later. Cytochrome P450 I (EROD activity) was decreased to 35-44% of control values by the ether anaesthesia and was restored to 80% of normal levels 2 h later. 5. Diether ether is known to be metabolized by cytochrome P450 IIE1 which is induced by fasting and by diethyl ether, and is possibly involved in the observed radical production, lipid peroxidation, and loss of cytochromes P-450.