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2.  Differential effects of antioxidants, steroids and other compounds on benzo(a)pyrene 3-hydroxylase activity in various tissues of rat. 
British Journal of Cancer  1979;40(1):105-112.
Antioxidants were found to inhibit the mixed-function oxidation of benzo(a)pyrene in several tissues of untreated and 3-methylcholanthrene-pretreated rats. The enzyme systems in the liver, kidney and stomach were much more susceptible to inhibition than those in the lung, adrenal, colon and small intestine. In all tissues except the stomach it was found that 3-methylcholanthrene pretreatment led to a decrease in inhibition of benzo(a)pyrene 3-hydroxylase activity. It is suggested that antioxidants exert their protective effect against cancer by inhibiting the formation of carcinogenic metabolites. Of the various steroids tested, only 17 beta-oestradiol and oestrone were significantly inhibitory in most tissues. Cholesterol was found to increase benzo(a)pyrene 3-hydroxylase activity in the gastrointestinal tract.
PMCID: PMC2009946  PMID: 454558
5.  Molecular mechanisms of SR 4233-induced hepatocyte toxicity under aerobic versus hypoxic conditions. 
British Journal of Cancer  1993;68(3):484-491.
SR 4233 (3-amino-1,2,4-benzotriazine-1,4-dioxide) is the lead compound of the benzotriazene-di-N oxides which are selectively toxic to tumour cells under hypoxic conditions. However much higher concentrations given to rats caused bone marrow toxicity and necrosis of the low oxygen Zone 3 part of the liver. In the following effects of SR 4233 on hepatocytes under hypoxic vs aerobic conditions have been compared. (1) SR 4233 did not affect hepatocyte viability (as determined by plasma membrane disruption) or glutathione levels under aerobic conditions. SR 4233 however induced cyanide-resistant respiration, an indicator of redox cycling mediated oxidative stress and became cytotoxic if hepatocyte catalase or glutathione reductase was inactivated. Glutathione oxidation occurred well before cytotoxicity ensued. Addition of ascorbate markedly enhanced SR 4233 cytotoxicity to these compromised hepatocytes. (2) In contrast, SR 4233 was highly toxic to hypoxic hepatocytes. Addition of ascorbate to enhance SR 4233 reduction also caused a marked increase in hepatocyte toxicity and an SR 4233 radical was detected with ESR spectroscopy. SR 4233 cellular reduction and toxicity was prevented with fructose or inhibitors of NADPH:cytochrome P-450 reductase. Inactivation of catalase or glutathione reductase had no effect on SR 4233 toxicity and hepatocyte GSH was not oxidised indicating oxidative stress did not occur during hypoxic SR 4233 hepatocyte toxicity. (3) The lack of SR 4233 cytotoxicity under aerobic conditions could probably be attributed to the detoxification of the SR 4233 radical by mitochondrial oxidation as SR 4233, but not its metabolite SR 4317 markedly increased state III and IV mitochondrial respiration in the presence of NADH. The increased respiration was inhibited by the respiratory inhibitors KCN and antimycin A but not by rotenone. Furthermore SR 4233 cytotoxicity under aerobic conditions was markedly increased by partially inhibiting hepatocytes respiration with cyanide but not rotenone.
PMCID: PMC1968404  PMID: 8394729
6.  Malignant hyperthermia susceptibility: cardiac histomorphometry of dogs and young and market-weight swine. 
The defect causing malignant hyperthermia has been proposed to involve cardiac as well as skeletal muscle. We tested the hypothesis that histomorphometric parameters for ventricular wall from malignant hyperthermia-susceptible swine and dogs were abnormal. Hearts were obtained from: mature dogs, age- and weight-matched young swine (89 +/- 15 days, 30 +/- 3 kg); and market-weight swine (102 +/- 10 kg). Using light microscopy, estimates were made for muscle nuclear dimensions and the volume-fraction of nuclei, sarcoplasm, blood vessels, and interstitial space. Cardiac maturation in both MH and normal swine was accompanied by decreased myocyte volume-fraction due to decreased nuclear volume-fraction and increased interstitial space volume-fraction. Sarcoplasm and vasculature volume-fraction were unchanged after maturation. Nuclear volume-fraction was slightly greater (p less than 0.05) in the right ventricle than the left for malignant hyperthermia and normal swine. Myocyte nuclear dimensions were generally similar among animals. Dogs and the oldest group of swine were not significantly different. Myocytes of all swine contained multiple nuclei, closely spaced in rows of 2 to 12. In contrast, most myocytes of mature dogs apparently contained one or two nuclei. Histomorphometric values were not significantly different between normal and malignant hyperthermia young swine and dogs. However, within the market-weight swine, volume-fraction for malignant hyperthermia myocytes and myocyte nuclei was decreased and interstitial space was increased compared to normal.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC1255273  PMID: 3567752
7.  Porcine malignant hyperthermia susceptibility: increased calcium-sequestering activity of skeletal muscle sarcoplasmic reticulum. 
This study tested the hypothesis that calcium-sequestration by isolated sarcoplasmic reticulum was abnormal in skeletal muscle of malignant hyperthermia-susceptible swine. A heavy sarcoplasmic reticulum fraction was isolated from malignant hyperthermia and control muscle using differential and density-gradient centrifugation. Prior to onset of malignant hyperthermia, calcium-sequestering activity (Vmax at 37 degrees C, mumol calcium/mg/min) was twofold increased in malignant hyperthermia sarcoplasmic reticulum compared to control sarcoplasmic reticulum (1.96 +/- 0.50 versus 4.00 +/- 0.87, P less than 0.01), although thermodynamic and kinetic properties of this activity were otherwise indistinguishable between groups. This increased activity of the malignant hyperthermia sarcoplasmic reticulum fraction was associated with twofold increased concentration of Ca-ATPase and calsequestrin protein. When a malignant hyperthermia-reaction developed, calcium-uptake was depressed to less than 5% of control values. These data indicate that malignant hyperthermia is not initiated due to a defect in the calcium-sequestration mechanism, however, loss of calcium-uptake activity occurring after the onset of malignant hyperthermia might result in the propagation and irreversibility of the malignant hyperthermia reaction.
PMCID: PMC1255221  PMID: 3742368
8.  Porcine malignant hyperthermia susceptibility: hypersensitive calcium-release mechanism of skeletal muscle sarcoplasmic reticulum. 
This study tested the hypothesis that calcium-release from sarcoplasmic reticulum isolated from malignant hyperthermia swine had abnormal concentration-dependency on release modulators. Halothane stimulated half-maximal calcium-release at similar concentrations for malignant hyperthermia and control sarcoplasmic reticulum (0.10 +/- 0.04 mM). However, concentrations causing half-maximal calcium-release were lower for malignant hyperthermia sarcoplasmic reticulum (P less than 0.001) by an order of magnitude for Ca2+ (28.1 +/- 8.3 versus 1.23 +/- 0.45 nM), adenosine triphosphate (0.33 +/- 0.09 versus 0.023 +/- 0.014 mM) and caffeine (7.79 +/- 1.56 versus 0.80 +/- 0.44 mM). Half-maximal inhibition by Mg2+ occurred at threefold higher concentrations for malignant hyperthermia sarcoplasmic reticulum (0.23 +/- 0.02 versus 0.78 +/- 0.17 mM). The Ca2+-sensitivity curves for calcium-release by sarcoplasmic reticulum isolated from heterozygotes for the malignant hyperthermia-defect were indistinguishable from the averages of the curves for controls and malignant hyperthermia-homozygotes. Results of this study suggest that malignant hyperthermia is initiated due to a hypersensitive calcium-release mechanism which is inherited in an autosomal, codominant pattern and may be diagnosed using calcium-release sensitivity-tests on isolated sarcoplasmic reticulum.
PMCID: PMC1255220  PMID: 3742367
9.  Free-radical-mediated DNA binding. 
Free-radical metabolites can be generated metabolically by a one-electron reductase-catalyzed reaction or a "peroxidase" catalyzed oxidation or by photoactivation of a wide variety of aromatic xenobiotics. Radicals may also be generated during lipid peroxidation. Some radicals can react with DNA or bind covalently or noncovalently as a dismutation product or as a dimer, trimer or polymeric product. Modification to the DNA can result in single-strand breaks, loss of template activity, and crosslinking. The binding can prevent enzymic digestion. In some cases, the radicals react with oxygen, resulting before conversion to DNA reactive oxygen species. Most radicals probably do not interact with DNA.
PMCID: PMC1568602  PMID: 3007090
10.  Canine malignant hyperthermia susceptibility: erythrocytic defects--osmotic fragility, glucose-6-phosphate dehydrogenase deficiency and abnormal Ca2+ homeostasis. 
Two dogs were diagnosed as malignant hyperthermia susceptible based on increased susceptibility (P less than 0.001) of biopsied muscle to caffeine-induced contracture. Erythrocytes from malignant hyperthermia and normal dogs were then examined for an antioxidant system deficiency. Values for serum muscle enzymes, reticulocytes and corpuscular hemoglobin were mildly elevated. Osmotic fragility was increased: hemolysis occurred at a NaCl concentration 10 mM higher than for normal dogs (P less than 0.001). A 35% glucose-6-phosphate dehydrogenase deficiency (P less than 0.001) with a 40% compensatory increase (P less than 0.01) in 6-phosphogluconate dehydrogenase activity was found. The membrane Ca2+-activated ATPase activity was abnormal: 100% increased with a 40% decreased Arrhenius activation energy (P less than 0.005) and increased thermostability. A 40% increased intracellular accumulation of total Ca2+ occurred in response to in vitro energy depletion in erythrocytes from one malignant hyperthermia dog (P less than 0.01). The multifactorial pattern of inheritance and the broad spectrum of malignant hyperthermia susceptibility are proposed to result from an antioxidant system deficit unmasking or aggravating an intrinsic muscle membrane anomaly. An individual from a family with a history of malignant hyperthermia or unexplained anesthetic death should be considered malignant hyperthermia susceptible if erythrocyte osmotic fragility is abnormal and there is a mild, unexplained elevation in serum creatine kinase.
PMCID: PMC1236089  PMID: 6150753
11.  Effect of chrysotile asbestos and silica on the microsomal metabolism of benzo(a)pyrene. 
The effects of chrysotile, water-leached chrysotile, and silica on microsomal metabolism of benzo(a)pyrene in vitro were studied. Examination of benzo(a)pyrene metabolites generated by 3-methylcholanthrene-pretreated rat liver microsomes, in the presence of chrysotile fibers, revealed a reduction in the overall metabolism of the hydrocarbon. Thus, chrysotile appeared to modify the activities of aryl hydrocarbon hydroxylase and epoxide hydrolase. Leaching chrysotile in deionized water for 24 hr elicited a similar response. Silica, in contrast to chrysotile, did not decrease the microsomal metabolism of benzo(a)pyrene. Chrysotile, as well as water-leached chrysotile, considerably diminished the microsomal production of water-soluble benzo(a)pyrene metabolites. Precoating the fibers with heparin or bovine serum albumin partially abolished this inhibition. The liver microsomal metabolism of benzo(a)pyrene in rats subjected to intraperitoneal administration of chrysotile was 58% of that in untreated rats.
PMCID: PMC1569290  PMID: 6315372
12.  Canine Malignant Hyperthermia: Diagnosis of Susceptibility in a Breeding Colony 
The Canadian Veterinary Journal  1983;24(6):172-177.
Fifteen related dogs were studied for susceptibility to malignant hyperthermia using halothane challenge and caffeine contracture tests. These dogs had hypertrophied muscles, were of a nervous temperament and had rectal temperatures at the upper limit of the normal range. Clinical pathology findings were mild elevations of serum aspartate transaminase and mean corpuscular hemoglobin. In vitro caffeine contracture tests were performed on muscle biopsies from five of these dogs. The concentration of caffeine required to increase resting tension by 1 g in biopsy specimens of these dogs was significantly lower than that required for control dogs: 7.6 ± 1.38 (x̄ ± SEM) versus 15.5 ± 2.52 mM (P < 0.025), and in the presence of 1% halothane, 3.6 ± 1.44 versus 10.6 ± 2.19 mM (P < 0.05). Internal nuclei, fiber caliber variation and fiber hypertrophy were found in histological studies of muscle biopsies. Two other dogs possibly died of a canine stress syndrome analagous to the porcine stress syndrome which occurs in malignant hyperthermia susceptible swine. Eight others of this family were anesthetized with halothane or methoxyflurane. Methoxyflurane did not trigger the syndrome. The first exposure to halothane caused death from malignant hyperthermia in two dogs and a third died on the second exposure to halothane. Postmortem findings were nonspecific. The other three dogs exposed to halothane recovered uneventfully. Inheritance of the defect conforms to a multifactorial pattern, with gradations of susceptibility.
PMCID: PMC1790357  PMID: 17422267
13.  Molecular mechanisms of tirapazamine (SR 4233, Win 59075)-induced hepatocyte toxicity under low oxygen concentrations. 
British Journal of Cancer  1995;71(4):780-785.
Previously we showed that tirapazamine (SR 4233, Win 59075) is cytotoxic towards hepatocytes under conditions of hypoxia but not in 10% or 95% oxygen and that bioreduction by DT-diaphorase or cytochrome P450 is not a major pathway. In the present study, we report that tirapazamine is highly cytotoxic to isolated rat hepatocytes maintained under 1% oxygen and the molecular cytotoxic mechanism has been elucidated. Cytotoxicity was prevented by the cytochrome P450 2E1 inhibitors phenyl imidazole, isoniazid, isopropanol or ethanol, suggesting that cytochrome P450 2E1 catalysed tirapazamine reductive bioactivation. By contrast, dicoumarol, a DT-diaphorase inhibitor, markedly increased tirapazamine-induced cytotoxicity. Cytotoxicity was also inhibited in normal but not DT-diaphorase-inactivated hepatocytes by increasing cellular NADH levels with lactate or ethanol or the mitochondrial respiratory inhibitors. Evidence that oxygen activation contributed to cytotoxicity was that glutathione oxidation occurred well before cytotoxicity ensued and that tirapazamine was more cytotoxic towards catalase- or glutathione reductase-inactivated hepatocytes. Furthermore, polyphenolic antioxidants such as quercetin, caffeic acid or purpurogallin, the radical trap Tempol or the iron chelator desferrioxamine prevented tirapazamine-mediated cytotoxicity. However, the antioxidants diphenylphenylenediamine, butylated hydroxyanisole or butylated hydroxytoluene did not prevent cytotoxicity and malonaldehyde formation was not increased, suggesting that lipid peroxidation was not important. The above results suggest that DT-diaphorase detoxifies tirapazamine whereas reduced cytochrome P450 reduces tirapazamine to a nitrogen oxide anion radical which forms cytotoxic reactive oxygen species as a result of redox cycling.
PMCID: PMC2033725  PMID: 7710944
14.  Effects of Ureaplasma diversum on bovine oviductal explants: quantitative measurement using a calmodulin assay. 
Calmodulin (CAM) acts as an intracellular regulator of calcium, an important mediator of many cell processes. We used the CAM assay and electron microscopy to investigate the effects of Ureaplasma diversum on bovine oviductal explants obtained aseptically from slaughtered cows. A stock suspension of U. diversum (treated specimens) and sterile broth (controls) was added to replicates of cultured explants and incubated at 38 degrees C in an atmosphere of 5.5% CO2 for 48 hours. Explants were examined for ciliary activity, extracellular CAM loss, and for histological and ultrastructural changes. Explants and their culture media were examined for changes in CAM concentration. All experiments were replicated three times. In addition, U. diversum, medium and broth were assayed for CAM content. The concentrations of CAM in explants and media changed significantly (p < 0.05) in samples which were inoculated with U. diversum when compared to controls. The controls and infected specimens did not differ histologically or ultrastructurally, but U. diversum was seen to be closely associated with infected explant tissue. In view of this close affinity it is assumed the loss of CAM from the oviductal cells was causally related, but this was not proven. The failure to show cell membrane injury on light and electron microscopic examination was probably related to the short duration of the experiment and may only point out the sensitivity of the CAM assay in detecting early cell membrane injury. Compromise in characteristics of the medium to support both, the viability of oviductal cells and U. diversum limited the experimental time to 48 hours.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC1263676  PMID: 8004536
15.  Blood volume increase in salt-induced pulmonary hypertension, heart failure and ascites in broiler and White Leghorn chickens. 
In this study we tested the hypothesis that excess dietary salt produces an expansion of extracellular fluid volume which may be associated with pulmonary hypertension-induced right ventricular failure in chickens with rapid growth rates. One-week-old broiler and White Leghorn chickens were given 0.5% salt in their drinking water for three weeks. Saline water had a minimal effect on White Leghorns. The hypothesis appears to be correct since salt-treatment in broilers resulted in up to 30% expansion in blood volume and there was 50% mortality from pulmonary hypertension-induced right ventricular failure and ascites. There was marked (up to 88% in some broilers) right ventricular hypertrophy, an indicator of pulmonary hypertension. There was less left ventricular hypertrophy as shown by an increase in the ratio of the right to total ventricle weight. There was up to 32% decrease in growth rate. There was renal hypertrophy in the salt-treated birds as shown by a higher kidney to body weight ratio.
PMCID: PMC1263603  PMID: 8490804
16.  Measurement of the cytotoxic effects of different strains of Mycoplasma equigenitalium on the equine uterine tube using a calmodulin assay. 
The cytopathic effects induced by five strains of Mycoplasma equigenitalium for cells of equine uterine tube explants were tested by measuring changes in cellular and extracellular concentrations of calmodulin (CaM). Calmodulin concentrations in samples of total homogenate (TH) and total homogenate supernates (THS) of the infected equine uterine tube explants were significantly lower than respective measurements on noninfected controls. In tissue culture medium fractions (TCM) of some infected explants, CaM concentrations were significantly higher than noninfected controls (p > 0.95). The results suggest that M. equigenitalium colonization on ciliated cells of the equine uterine tube can affect the permeability of the cell membrane leading to leakage or release of CaM during cell breakdown. Measurement of CaM concentrations in samples of TH revealed significant differences in the cytotoxic effects induced by different strains of M. equigenitalium on the equine uterine tube (EUT). The data suggests that some strains of M. equigenitalium may have a role in reproductive failure in the mare. In addition comparisons of the means of the concentrations of CaM in samples of TH or THS in EUT explants from four mares in the follicular and four in the luteal phase of the estrous cycle were found to be not significantly different.
PMCID: PMC1263565  PMID: 1477802
17.  Malignant hyperthermia susceptibility: biochemical basis for pathogenesis and diagnosis. 
Malignant hyperthermia (MH) is a hypermetabolic and hypercontractile syndrome triggered by anesthesia or various stressors that cause a sustained increase in sarcoplasmic ionized Ca. Susceptibility is apparently inherited in an autosomal dominant pattern. The primary molecular defect results in hypersensitive ligand-gating of the Ca-release channel of sarcoplasmic reticulum (SR) in skeletal muscle: channel opening is stimulated by abnormally low concentrations of agonist. We attribute MH to a mutation in the gene for the fast twitch muscle isoform of the Ca-channel, resulting in the expression of a cardiac-like isoform in fast muscle. Syndromes with some resemblance to MH can occur due to other genetic or acquired imbalances in Ca-flux across SR that favor net release of Ca. Either defective uptake or release can be detected as increased sensitivity of muscle to the contracture-producing effects of caffeine and halothane. Thus, caffeine and/or halothane contracture tests for MH-susceptibility may give false positives when there is decreased Ca-uptake, such as in muscular dystrophies. Ca-channel hypersensitivity and decreased Ca-uptake activity can be detected by assays using isolated SR. Functional assays using lymphocytes are being assessed as potential replacements for muscle contracture tests. Polymorphism analysis of proteins or nucleic acids for the MH or closely-linked genes has been used to trace the inheritance of MH-susceptibility.
PMCID: PMC1255610  PMID: 2155050

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