Relationship between the depletion of O6-methylguanine-DNA methyltransferase by O6-methylguanine and the stimulation of DNA synthesis and growth of cultured chick hepatocytes.

O6-Methylguanine-DNA methyltransferase (O6-MT) has been described as a DNA repair enzyme that reverses alkylation damage at the O6 position of guanine in DNA. We demonstrate that the concentration of this protein decreases immediately prior to DNA synthesis in cultured chick hepatocytes. If intracellular levels are experimentally depleted by treatment of cultures with O6-methylguanine, DNA synthesis occurs as an associated resultant. This effect is dose dependent and can be followed by discernible morphological changes of organoids in culture. Increased and altered growth caused by O6-methylguanine was quantified and was also found to be dose dependent. Therefore, O6-MT may play a role in the regulation of DNA synthesis.

Pretreatment with paracetamol inhibits metabolism of enflurane in rats.

We studied the interaction between paracetamol (acetaminophen U.S.P.) and enflurane. Sixteen rats were assigned to four groups (n = 4) to receive: paracetamol 7.5 mg/100 g body weight; paracetamol plus 1% enflurane; 1% enflurane alone, or no treatment (controls). Animals were killed 6 h later. A second series of 16 were treated identically, but were killed after 24 h. Measurements were made of fluoride concentrations in serum, liver and urine (indicators of biotransformation of enflurane), paracetamol concentrations in urine, pathological changes in liver samples, and concentrations of the enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum. Pretreatment with paracetamol significantly decreased urinary fluoride at 6 and 24 h after exposure to enflurane, but decreased fluoride concentrations in serum and liver only at 6 h after exposure to enflurane. Paracetamol concentrations in urine did not change after exposure to enflurane. Exposure to paracetamol alone increased AST and ALT. At 24 h after exposure to enflurane, serum concentrations of enzymes in rats pretreated with paracetamol were similar to those of control rats. Pretreatment with paracetamol may therefore inhibit metabolism of enflurane. Although no hepatic damage was observed, the increased in AST and ALT suggested subclinical liver damage in rats given only paracetamol.

Aroclor 1254 treatment and fasting influences on rat liver mitochondrial carbamoyl phosphate synthesis with ADP and ATP.

Previous studies have shown that the polychlorinated biphenyl mixture, Aroclor 1254 (ARO), -induced wasting in male rats is associated with increased permeability of hepatic mitochondria. This was correlated with hyperuremia and stimulated urea synthesis, hypoglycemia and suppressed glucogenesis after an ammonium acetate injection, and decreased retention of assimilated nitrogen and food intake. For ARO-toxic rats (100 mg/kg, ip, for 1, 2, and 4 days) versus Tween 80-treated, ad libitum-fed controls, mitochondrial carbamoyl phosphate (CP) formation (the initial step in urea synthesis from NH4+) was progressively stimulated for the duration of treatment from NH4+ and ATP but not from NH4+ and ADP. ARO maximal stimulation of CP formation also correlated with significant loss in body weight. Mitochondrial ornithine transcarbamoylase synthesis of citrulline from ornithine and carbamoyl phosphate was also stimulated. In comparison to fasted rats (24 hr), mitochondrial CP synthesis from NH4+ was enhanced with ADP but not with ATP. This ARO uncoupling of mitochondrial NH4+ metabolism and stimulation of CP formation with exogenous ATP and citrulline synthesis may have resulted from increased availability of substrates and cofactors in the matrix space, leakage of enzymes from the matrix, or a combination of these effects. These results are consistent with an increased inner membrane permeability and fragility during isolation and assays. In agreement with our previous studies, the data show that ARO exposure poises hepatic mitochondria toward the synthesis of urea intermediates.

Plasma lidocaine concentrations in conscious horses after cervicothoracic (stellate) ganglion block with 1% lidocaine HCl solution.

Arterial and/or central venous plasma concentrations of lidocaine were determined in 12 nonmedicated adult horses (422 +/- 59 kg of body weight, mean +/- SD) after injecting a 1% lidocaine HCl solution into the cervicothoracic ganglion (CTG). A mean dosage of 2.9 +/- 0.5 mg of lidocaine/kg of body weight was used to induce unilateral CTG blockade in 8 horses and 4.8 +/- 0.8 mg was used to induce bilateral CTG blockade in 4 horses. Blood samples were collected before and at 5, 15, 30, 45, 60, 75, 90, 105, and 120 minutes after injection. The plasma lidocaine concentrations were determined by use of gas chromatography (sensitivity less than 0.01 microgram/ml). Cervicothoracic sympathetic blockade was characterized by Horner's syndrome and by profuse sweating over the face, neck, and thoracic limbs. Mean maximal venous concentrations of lidocaine were 0.86 +/- 0.33 microgram/ml at 26.3 +/- 6.9 minutes after unilateral CTG blockade, and 1.14 +/- 0.25 micrograms/ml at 31.2 +/- 18.9 minutes after bilateral CTG blockade. The mean venous and arterial concentrations of lidocaine were not significantly different at 45 and 120 minutes after injection. Venous concentrations of lidocaine were consistently higher than were concentrations in simultaneously collected arterial blood samples in 2 horses in which the right CTG and brachial plexus were temporarily anesthetized after repeated administration of 100 ml of lidocaine into the right CTG.(ABSTRACT TRUNCATED AT 250 WORDS)

Toxicity of chlorine dioxide in drinking water.

Chlorine dioxide (ClO2) is currently being considered as an alternate to chlorine as a disinfectant for public water supplies. Studies were conducted to determine the toxicity of ClO2 (0, 1, 10, 100, 1000 mg/l) and its metabolites, ClO3- and ClO3- (10, 100 mg/l) in drinking water in rats. After nine months treatment the osmotic fragility of the red blood cells was decreased in all treatment groups, while a decreased blood glutathione was only observed in the metabolite groups. At 2, 4 and 6 mon no significant hematologic changes were noted in treated rats compared to control. However, after 9 mon RBC counts, hematocrit and hemoglobin were decreased in all treatment groups. ClO2, ClO2- and ClO3- administered chronically in drinking water for three months inhibited the incorporation of 3H-thymidine into nuclei of rat testes. Also, this inhibition was observed in the liver of ClO2- groups and in the kidney of 100 mg/l ClO2- treatment. The incorporation in small intestinal nuclei was increased in both 10 and 100 mg/l ClO2 and in 10 mg/l ClO2-. The treatment with Cl-compounds decreased rat body weight in all groups after 10 and 11 months treatment.

The kinetics of chlorite and chlorate in rats.

Chlorine dioxide (ClO2) is under consideration as an alternative to chlorination as a disinfectant for public water supplies. The primary products resulting from ClO2 disinfection of surface waters are chlorite (ClO2-) and chlorates (ClO3-). The kinetics of 36ClO2- and 36ClO3- was studied in rats. Radioactivity was rapidly absorbed from the gastrointestinal tract following the administration of (0.17 microCi) 36ClO2- or (0.85 microCi) 36ClO3- orally; and 36Cl in plasma reached a peak at 2 hr and 1 hr, respectively. After 72 hr, radioactivity was highest in whole blood, followed by packed cells, plasma, stomach, testes, skin, lung, kidney, duodenum, carcass, spleen, ileum, brain, bone marrow and liver in 36ClO2- treatment. 36Cl excretion was greatest at 24 hr after the administration of 36ClO3-, but in the 36ClO2-, the excretion most likely represented saturation of the biotransformation and excretion pathways. About 40% of the total initial dose was excreted at 72 hr in the urine and feces in both treatments. No 36Cl was detected in expired air throughout the 72 hr studied.

The effects of selenium on the emergence of aflatoxin B1-induced enzyme-altered foci in rat liver.

The effects of selenium on the emergence of aflatoxin B1 (AFB1)-induced enzyme-altered foci were studied in male Sprague-Dawley rats. Animals were fed a selenium-deficient diet and supplemented with 5.0, 2.0, and 0.2, or 0 ppm selenium in drinking water for 3 weeks prior to initiation with 2.0 mumol/kg AFB1. After a 1-week period of selenium normalization, the animals were placed on a diet of ordinary rat chow, and were administered a promoting regimen of 500 ppm phenobarbital in drinking water for 1 week, after which time each rat received a two-thirds partial hepatectomy. The promoting regimen of phenobarbital in tap water was then reduced to 100 ppm and continued for 7 weeks. Subsequently, the rats were sacrificed, their livers excised, and fresh frozen sections prepared and stained histochemically to demonstrate areas of gamma-glutamyl transpeptidase (GGT) activity. Selenium supplementation was observed to diminish the induction of GGT-positive foci, especially at the 5.0-ppm level. These data suggest that selenium is able to protect against the hepatocarcinogenic effects of AFB1 in the rat, and that the enzyme-altered foci bioassay may be a useful technique in assessing the interaction of selenium on the process of hepatocarcinogenesis.

Decreased enkephalinase activity following postnatal treatment with phenobarbital.

The effect of postnatal administration of phenobarbital on enzymes degrading enkephalin was examined. Daily subcutaneous injections (45 mg/kg) of phenobarbital were given to male and female rats from postnatal day 1 to 19. Brains from rats treated with saline and phenobarbital were used to prepare aminopeptidases (high speed supernatant) and enkephalinase A (synaptosomal membrane preparation). Incubation of methionine enkephaline (ME) with aminopeptidases from rat brain liberated tyrosine (T), while incubating with enkephalinase A resulted in the formation of tyrosylglycylglycin (TGG). Separation and quantification of tyrosine, tyrosylglycylglycin and methionine enkephalin was performed using a high performance liquid chromatograph, coupled to electrochemical and ultraviolet detectors in series. The treatment of the rats with phenobarbital resulted in a significant inhibition of enkephalinase A when measured in vitro, using methionine enkephalin as substrate. Preliminary studies with secobarbital show similar results to those obtained with phenobarbital.

Hexacarbon neuropathy: tracking a toxin.

This paper will present the progression of hexacarbon studies which followed the investigation of circumstances surrounding the occurrence of peripheral neuropathy in an occupational setting. The identification of methyl n-butyl ketone as the neurotoxin led to systematic studies of the biotransformation and mechanism of neurotoxicity. A consistent line of evidence showed that the neurotoxic potential of hexacarbons is directly related to the formation of the gamma-diketone metabolite, 2,5-hexanedione. The precise nature of the chemical interaction at the neurofilament is the subject of continuing investigation.

The effects of the quinone type drugs on hydroxyl radical (OH.) production by rat liver microsomes.

The quinone drugs are known to be metabolized to semiquinone free-radical intermediates and to enhance NADPH oxidation in microsomal system. The effect of adriamycin and mitomycin C on the decarboxylation of [14C] carboxyl benzoate via hydroxyl radical (OH.) production in the microsomal system was examined. The activity of these drugs was compared to 5-fluorouracil, cyclophosphamide, and methotrexate, which are inactive in oxygen consumption experiments and are non-quinone-type drugs. Adriamycin and mitomycin C stimulated decarboxylation of benzoate 100 and 50% above the controls, respectively, while 5-fluorouracil, cyclophosphamide, and methotrexate were not different from controls. Addition of superoxide dismutase increased benzoate decarboxylation with or without the drugs present, while catalase was inhibitory in both circumstances. These results suggest that the quinone drugs enhanced hydroxyl radical (OH.) production by liver microsomes, and offer a possible mechanism of cellular toxicity by these agents.

Trichloroethylene effects on the formation of enzyme-altered foci in rat liver.

The initiating and promoting effects of trichloroethylene in rat liver were investigated using the enzyme-altered foci bioassay. The incidence of gamma-glutamyl transpeptidase (GGT)-positive foci was used as an early histochemical marker of putative preneoplastic hepatocytes. A single PO dose of trichloroethylene (490 mg/kg) was administered in corn oil to rats which had been partially hepatectomized 24 h previously. Three days following gavage with the chlorinated hydrocarbon the rats were promoted with an 8-week regimen of 500 ppm phenobarbital in drinking water. This protocol is known to induce enzyme-altered foci in the livers of animals which have received an initiating dose of a genotoxic carcinogen. Trichloroethylene was not found to induce GGT-positive foci under these conditions. Additionally, groups of rats were partially hepatectomized, initiated with N-nitrosodiethylamine (30 mg/kg; PO) and administered five times weekly doses of 200 mg trichloroethylene per rat in order to investigate the promoting activity of the chlorinated hydrocarbon in rat liver. No significant promoter effects were observed with trichloroethylene, although the results in this case were somewhat equivocal. The findings of these investigations are taken as partially supportive of an epigenetic, cytotoxic mechanism of tumorigenic action of trichloroethylene.

An electrophysiological study of 2-hexanone and 2,5-hexanedione neurotoxicity in rats.

n-Hexane and its metabolites are neurotoxic to animals and man. Studies have revealed a progressive neuropathy which affects the distal regions of motor and sensory peripheral nerves. This paper describes efforts to determine whether 2-hexanone or 2,5-hexanedione is more neurotoxic than 2-hexanone and that it first affects the distal axon. Concentrations of 20 mM produced no effects after 3 weeks but 40 mM increased distal latency after 2 weeks.

Binding of chloroform to the cysteine of hemoglobin.

The products of the covalent binding of chloroform to rat hemoglobin during microsomal metabolism were isolated and identified by gas chromatography (GC) and mass spectroscopy (MS). After isolation by Proteinase K hydrolysis, amino acid analysis and cellulose thin-layer chromatography (TLC), the major product was identified by GC/MS as N-hydroxymethyl cysteine and a minor product as 2-hydroxythiazolidine-4-carboxylic acid. N-Hydroxymethyl cysteine is proposed to be formed during isolation from the 2-oxothiazolidine-4-carboxylic acid present in the intact hemoglobin.

Prenatal alcohol exposure alters enkephalin levels, without affecting ethanol preference.

Pregnant dams were pair-fed a liquid diet containing 35% ethanol derived calories or isocaloric sucrose during the last two trimesters of pregnancy. No differences were observed in adult ethanol preference between fetal alcohol exposed (FAE) animals and pair-fed controls. However, Met- and Leu-enkephalin levels were significantly elevated in globus pallidus of adult FAE animals. Pituitary levels were unaffected.

Analysis of enkephalins, beta-endorphins and small peptides in their sequences by highly sensitive high-performance liquid chromatography with electrochemical detection: implications in opioid peptide metabolism.

Sensitivity in the 10-100 pg range for enkephalins, beta-endorphin, tyrosine (T), 12 tyrosylglycine (T-G) and tyrosylglycylglycine (T-G-G) was attained by using a high-performance liquid chromatographic (HPLC) method with electrochemical detection which is at least 100 times more sensitive than HPLC with UV detection. The chromatographic conditions on a reversed-phase C18 silica column were 50 mM sodium phosphate buffer (pH 2.1) (A) in acetonitrile-methanol (1:1) (B), isocratic mixture, flow-rate 0.6-1 ml/min, UV detection at 205 nm, electrochemical oxidation potential + 1.25 V. The separation of T, T-G and T-G-G was obtained by using 10% B while the separation of the pentapeptide, enkephalins required 40% B. Separation of enkephalins from beta-endorphin was attained at a shorter retention times did not exceed 15 min. This method can be used to determine tissue levels and pharmacodynamics of enkephalins and beta-endorphin. A highly specific measurement of the different enzymes involved in the metabolism of enkephalin has been achieved.

Prolonged intermittent footshock stress decreases Met and Leu enkephalin levels in brain with concomitant decreases in pain threshold.

The influence of a 21 day intermittent footshock regimen upon enkephalin levels in brain and adrenals was examined in the rat. Changes in pain sensitivity as well as analgesic and hyperthermic responsiveness to morphine (7.5 mg/kg) were also monitored. Following the stress regimen, Met and Leu enkephalin levels were decreased by 40 to 50% in brain, but were unchanged in adrenals. Post-stress pain thresholds were markedly decreased in stressed animals while the analgesic properties of morphine were enhanced. Core body temperature of stressed animals was significantly raised, but the hyperthermic response to morphine was unchanged.

Dexamethasone and stress-induced analgesia.

Analgesia induced in rats by cold-water swim stress and measured by the tail-flick and hot-plate methods was significantly antagonized after IP pretreatment for 3 days with 8 mg/kg dexamethasone. The analgesia developed by the cold-water swim stressor was also attenuated by 1 mg/kg naloxone. These results suggest that the corticosteroids may have a role in modulating stress-induced analgesia and that the adrenal-pituitary axis modulates the endogenous opiate system. These conclusions are based on recent reports that indicate the release of the opiate-like peptide beta-endorphin and adrenocorticotropin (ACTH) from the pituitary are increased by acute stress and inhibited by administration of the synthetic glucocorticoid dexamethasone.

Metabolism and pharmacokinetics of alternate drinking water disinfectants.

The chlorination of surface waters is known to elevate trihalomethanes; consequently, chlorine dioxide (ClO2) is being considered as an alternative disinfectant. The primary products resulting from ClO2 disinfection of waters are chlorites (ClO2-) and chlorates (ClO3-). Studies in rats revealed that ClO2 is converted to chloride (Cl-), ClO2- and ClO3-. ClO2- and ClO3- are excreted as Cl-, ClO2- and Cl-, ClO2-, ClO3-, respectively. Radioactivity was rapidly absorbed from the gastrointestinal tract following the administration of 36ClO2 orally, and the half-life for the elimination of 36Cl from the rat was 44 hr, corresponding to a rate constant of 0.016/hr. After 72 hr, radioactivity was highest in plasma, followed by kidney, lung, and stomach. 36Cl in plasma reached a peak at 2 hr and 1 hr after oral administration of 36ClO2- and 36ClO3-, respectively. 36Cl excretion was greatest 24 hr after the administration of 36ClO3-, but in the case of 36ClO2-, the excretion probably represented saturation of the biotransformation and excretion pathway. A low activity in packed cells compared to plasma was detected in chlorate ingestion, rather than an even distribution in chlorite treatment. Chloroform determinations in rat blood after one year indicated that chloroform was significantly higher than control in the 100 and 1000 mg/l. ClO2 groups. However, no significant values were observed in the 1 or 10 mg/l. ClO2 and ClO2 metabolites groups. ClO2 and its metabolites are eliminated from the body more rapidly than chlorine, and they do not appear to increase trihalomethane concentrations at low dosages.

Assessment of maternal toxicity, embryotoxicity and teratogenic potential of sodium chlorite in Sprague-Dawley rats.

Groups of up to 13 pregnant rats were individually caged. Body weight, food and water consumption were recorded at days 1, 8, 15 and 22 of gestation and the dams were treated on days 8-15 with sodium chlorite, 0.1%, 0.5% or 2% in drinking water or by injection of 10, 20, or 50 mg/kg IP or by gavaging with 200 mg/kg. To prevent ingestion of stillborn pups some dams were sacrificed at day 22. Other dams were allowed to deliver at term. Fetuses were weighed, measured and examined for soft tissue and skeletal malformations. Sodium chlorite, 20 or 50 mg/kg daily IP or gavaging with 200 mg/kg, caused vaginal and urethral bleeding. Doses of 10, 20 or 50 mg/kg daily IP caused 0, 50 and 100% mortality of dams, respectively. No deaths were caused by sodium chlorite in the drinking water, but the dams' body weight, water and food consumption decreased during all treatments except 0.1% in the drinking water. Blood smears from the dams injected IP or drinking 2% sodium chlorite showed irregular, bizarre and ruptured erythrocytes. Injection of 10 or 20 mg/kg or drinking 2% resulted in decreased litter size and increased stillbirths and resorption sites. Drinking 0.1% or 0.5% sodium chlorite did not produce any significant embryotoxicity. With all treatments, no significant gross soft tissue or skeletal malformations were observed. Postnatal growth of the pups was not affected by any treatment of the dams during the gestation period.