Sublethal effects of treated liquid effluent from a petroleum refinery. I. Chronic toxicity to flagfish

Continuous exposure to 28% refinery effluent caused severe sublethal damage to flagfish (Jordanella floridae), including smaller size of first- and second-generation fish, hemorrhaging of fins in all fish, spinal curvature in the second generation, as well as delayed and less frequent spawning with fewer eggs per spawn, compared to the controls. Effluent at 0.92% caused no deleterious effects. The threshold for sublethal effect was approximately 9%, which caused only one deletrious effect of questionable overall importance, out of 15 major items assessed. The sublethal findings are considered representative for a well-treated refinery effluent; this one had average values for ammonia, pH, and oil-plus-grease that were close to or within Canadian regulations, sulphide and phenol levels that were lower than the regulation, and total residue somewhat higher than regulations. For the 30 twice-weekly samples used in this experiment, mortality of rainbow trout (Salmo gairdneri) in full-strength effluent averaged only 5% and no single sample caused > 50% mortality. Treatment of refinery effluent to meet Canadian regulations would mean that at most refinery sites, effluent would be diluted to the no-effect levels found in this experiment.     

Liver microsomal enzyme induction and toxicity studies with 2,4,5,3',4'-pentabromobiphenyl

A relatively minor component of Firemaster, 2,4,5,3′,4′-pentabromobiphenyl (PenBB), was purified and compared to the effects of Firemaster and 3,4,5,3′,4′,5′-hexabromobiphenyl (HBB). Male rats were given 90 mg/kg, ip, of PenBB or Firemaster and killed 2 weeks later. Blood samples were clinically analyzed and various tissues were examined for pathological effects. Induction of hepatic microsomal drug metabolizing enzymes and effects on the immune system were also investigated. No hematologic changes could be detected 2 weeks after either treatment. PenBB caused a significant loss in body weight, and the spleen weight to body weight and thymus weight to body weight ratios were slightly decreased by this treatment. Liver weight to body weight ratios were increased by both treatments. Ultrastructural changes in hepatocytes included proliferation of the smooth endoplasmic reticulum and the appearance of lipid droplets and concentric membrane whorls. The splenic T helper and B cells were substantially impaired by Firemaster and particularly by PenBB. There was a marked increase in liver microsomal cytochrome P-450 content and its CO difference spectrum absorption maximum was shifted to 448.5 nm. The microsomal ethyl isocyanide difference spectrum $\text{455}\text{430}\text{-nm}$ ratio was also increased. Benzo[a]pyrene hydroxylase, p-nitrophenol UDP-glucuronyltransferase, and epoxide hydrolase activities were markedly increased. Less pronounced induction of NADPH-cytochrome P-450 reductase, and aminopyrine-N-demethylase activities occurred. These results, in addition to the results of SDS-polyacrylamide gel electrophoresis suggest that the mono-ortho-brominated congener is predominantly a 3-methylcholanthrene (MC)-like inducer and is toxic. In another experiment when enzyme induction by PenBB (30 mg/kg) was compared to that caused by 30 or 2 mg/kg of HBB it was evident that the non-ortho-substituted congener is the more potent MC-like inducer. Therefore, we conclude that the single ortho halogen of PenBB may weaken but not abolish its toxicity and MC-like enzyme induction effects.     

Effect of a single oral dose of malathion on D-glucose and glycine uptake and on brush border enzymes in rat intestine

The effect of a single oral dose of malathion (1 g/kg body wt.) on the digestive and absorptive functions of the intestinal epithelium has been investigated in rats. The absorption of glucose and glycine was considerably reduced (35%) in pesticide fed animals compared to controls. The activities of brush border sucrase, lactase, alkaline phosphatase, Mg²⁺-ATPase and lactate dehydrogenase were also significantly depressed in malathion exposed rats, but there was no change in the leucine aminopeptidase levels under these conditions. Mucosal DNA, RNA and protein contents remained unaltered in pesticide toxicity. These results suggest that malathion toxicity induces functional derangements of the intestine.     

In vivo studies on the target tissue metabolism,covalent binding,glutathione depletion,and toxicity of 4-ipomeanol in birds,species deficient in pulmonary enzymes for metabolic activation

Comparative studies on the target organ for covalent binding and toxicity by 4-ipomeanol, a furan derivative metabolically activated by and toxic to the nonciliated bronchiolar epithelial (Clara) cells in mammalian lung, were performed in birds. In birds, whose lungs lack the typical pulmonary ciliated and nonciliated bronchiolar cells and are deficient in enzymes necessary for the metabolic activation of 4-ipomeanol, the target organ for toxicity and covalent binding by 4-ipomeanol was the liver. Tissue damage was not observed in lungs or kidneys of either Japanese quail or roosters at any dose of 4-ipomeanol tested. Likewise, the levels of irreversibly bound 4-ipomeanol metabolites were highest in liver, with much lower levels in lungs, kidneys, and all other organs studied. In addition, toxic doses of 4-ipomeanol markedly depleted hepatic, but not pulmonary or renal glutathione in Japanese quail. The covalent binding of 4-ipomeanol metabolites and concomitant depletion of tissue glutathione in the liver were both time and dose dependent. In quail, over a dose range of 5 to 75 mg/kg, no dose threshold for covalent binding or hepatic damage which depended upon substantial glutathione depletion was observed. Pretreatment of quail with phenobarbital had no effect on liver cytochrome P-450 levels, nor did it have any effect on tissue covalent binding or toxicity by 4-ipomeanol. Although 3-methylcholanthrene pretreatment nearly tripled the microsomal cytochrome P-450 levels in quail liver, it had relatively little or no effect on the hepatic covalent binding or toxicity by 4-ipomeanol. Prior treatment with the drug metabolism inhibitor, piperonyl butoxide, markedly decreased the covalent binding, glutathione depletion, and the toxicity of 4-ipomeanol. Treatment of quail with diethylmaleate produced a dose-dependent depletion of hepatic, renal, and pulmonary glutathione, and it markedly increased hepatic covalent binding by 4-ipomeanol, but had no effect on renal or pulmonary covalent binding. Diethyl maleate markedly enhanced the hepatic necrosis by 4-ipomeanol. The results are consistent with the view that 4-ipomeanol is metabolized preferentially in quail liver to highly reactive metabolites which can be detoxified by glutathione. It appears unlikely however, that differences in detoxification via glutathione are the primary determinants of the remarkable tissue selectivity for covalent binding and toxicity of 4-ipomeanol in different animal species in vivo. The present studies support the view that the tissue selectivity of 4-ipomeanol resides primarily in the ability of the target tissue to rapidly metabolize 4-ipomeanol to its ultimate toxic metabolite(s). These investigations also suggest that the bird may be a potentially useful animal model in studies on the relationship of target organ toxicity to the formation of reactive metabolites by other chemicals requiring metabolic activation.     

Biochemical changes in the liver of mice fed Mirex

In a study of the hepatic effects of Mirex (dodecachloroctahydro-1,3,4-methano-2H-cyclobuta[cd]pentalene), male Charles River CD-1 mice were fed 1–90 ppm Mirex in the diet for periods ranging from 1 to 70 wk. At regular intervals, heavy mitochondrial, light mitochondrial, microsomal, and soluble fractions were prepared from livers of three to six animals per group. Measurements of relative liver weight, DNA, protein, respiration, respiratory control, mitochondrial membrane permeability, mixed function oxidases (N-demethylation, aromatic hydroxylation, and cytochrome P-450), glucose-6-phosphatase, diene conjugates, and protein binding were carried out. Mirex caused an increase in relative liver weight, total liver DNA, total protein in each liver fraction, mitochondrial respiration and coupling, and mixed function oxidases. Except for DNA, each parameter increased with an increase in the level of Mirex in the diet and the duration of feeding. DNA was stimulated at all doses to 130–150% of control levels. A further rise in DNA occurred during the onset of the development of liver nodules. The action of Mirex on mitochondria occurred predominantly in the light mitochondrial fraction and resulted in a stimulation of respiration, increased coupling and a decrease in membrane permeability. Mirex added to control mitochondria (in vitro) produced none of these changes. Glucose-6-phosphatase activity was decreased with increasing levels of Mirex in the diet and duration of feeding. The concentration of microsomal diene conjugates was not affected by Mirex. Mirex was formly bound to soluble liver proteins both in vivo and in vitro, but did not displace cortisol from the cortisol receptor protein.     

Serum ferritin,gammaglutamyl-transferase and alcohol consumption in healthy middle-aged men

Serum ferritin was analysed in 169 middle-aged men. Ninety-one were heavy drinkers with increased serum gammaglutamyltransferase (GGT) values in otherwise normal routine screening tests; 39 were from the same birth year cohorts with normal GGT values; and 39 were teetotalers. Increased ferritin values were found in 67% of the heavy drinkers, but only in 2.5% in the two other groups. The mechanisms of the serum ferritin elevation in otherwise healthy alcohol over-consumers are unknown, but heavy drinking should be taken into consideration in the interpretation of increased serum ferritin values in individual cases.     

Pardachirus marmoratus (Red Sea flatfish) secretion and its isolated toxic fraction pardaxin: the relationship between hemolysis and ATPase inhibition

The ichthyocrinotoxic secretion of the Red Sea flatfish Pardachirus marmoratus and paradaxin, a polypeptide isolated from this secretion, were toxic when injected i.v. into rats. Pardaxin inhibits [Na⁺, K⁺] ATPase (Primoret al., 1980) but enhances esterase activity. Pardaxin caused hemolysis in dog red blood cells which lack [Na⁺, K⁺] ATPase. It is concluded that pardaxin induced hemolysis is not caused by [Na⁺, K⁺] ATPase inhibition.     

Toxicologic studies of four fluorescent whitening agents

Studies have been conducted to define the toxicologic properties of sodium 2-(4-styryl-3-sulfophenyl)-2H-naphtho-[1,2 d]triazole (I), disodium 4,4′-bis[(4-anilino-6-morpholino-1,3,5-triazin-2-yl)amino]stilbene-2,2′-disulfonate (II), disodium 4,4′-bis{[4-anilino-6-6-(N-methyl-2-hydroxyethylamino)-1,3,5-triazin-2-yl]amino}stilbene-2,2′-disulfonate (III) and disodium 4,4′-bis(2-sulfostyryl)biphenyl (IV). The following studies were performed on all the above materials: acute oral LD50, acute eye irritation, acute dust inhalation, acute fish toxicity, dominant lethal mutagenicity, teratogenicity, 90-day subacute oral toxicity in rats and dogs, and repeated insult patch tests in humans. Under the conditions of these studies the compounds were of low toxicity and were neither teratogenic nor mutagenic. Dose-related effects ranging from mild gastritis at 400 ppm to debilitation and peritonitis at 2000 ppm were observed in the 90-day dog feeding study with unbuffered compound I. Repetition of the study at 2000 ppm using compound I neutralized to pH 7 with hydrochloric acid showed no such effects. No adverse effects were observed with the unbuffered material in rats at levels up to 5000 ppm or in rhesus monkeys at a dose level of 50 mg/kg/day (approximately 2000 ppm). With compounds II, III and IV no effects were noted at levels up to 10,000 ppm in dogs or 5000 ppm in rats. No adverse effects have been observed at the end of 9 months of projected 2-year studies with compounds I, II, III and IV, at dietary levels up to 1000 ppm in rats and 2000 ppm in dogs, using neutralized material in the case of compound I in dogs. None of the materials exerted adverse effects in the first generation of a 3-generation rat reproduction study at dietary levels up to 1000 ppm. All were shown to be devoid of teratogenic activity in rabbits at dose levels of 10 and 30 mg/kg and of mutagenic activity in male mice. No evidence was obtained to indicate that any of the materials are sensitizing or phototoxic agents in either experimental animals or in man.     

Synergistic interaction between acivicin (AT-125) and 6-thioguanine in the murine leukemia L1210. Biochemical and cytokinetic considerations

The effect of the purine antagonist acivicin (AT-125) on the metabolism and cytotoxicity of 6-thioguanine was examined in the murine leukemia L1210. Cells exposed to 5 X 10(-6) M acivicin for 18 hr followed by 10(-5) M [14C]-6-thioguanine for 2 hr accumulated 2.90 +/- 0.17 nmoles 6-thioguanine/10(6) cells compared to 0.69 +/- 0.07 nmoles 6-thioguanine/10(6) cells in untreated controls. Intracellular accumulation of 6-thioguanine monophosphate, a lethal 6-thioguanine metabolite, increased from 0.27 +/- 0.05 to 1.08 +/- 0.13 nmoles 6-thioguanine monophosphate/10(6) cells following the same acivicin exposure. A similar increment was observed for the formation of 6-thioguanine triphosphate. These alterations in 6-thioguanine metabolism were associated with an increase in the intracellular level of 5-phosphoribosyl-1-pyrophosphate, an obligatory substrate in 6-thioguanine activation (57.9 +/- 7.6 vs 13.4 +/- 2.3 ng 5-phosphoribosyl-1-pyrophosphate/10(6) cells). In contrast, there was a 50% reduction in the amount of 6-thioguanine incorporated into RNA and DNA following acivicin pretreatment. Cytofluorometric analysis revealed that an 18-hr exposure to 5 X 10(6) M acivicin increased the population 5-phase cells, which are more sensitive to the actions of 6-thioguanine, by 50% relative to untreated controls. In both suspension culture growth and soft agar studies, the sequential administration of acivicin followed by 6-thioguanine resulted in substantial growth inhibitory activity; in contrast, the effects of the reverse sequence were subadditive. Pretreatment of L1210 cells with acivicin potentiates the action of subsequently administered 6-thioguanine, and the mechanism may involve both biochemical as well as cytokinetic factors. In vivo studies involving the sequential administration of these agents appear warranted.     

Separation of prostaglandin E1 from its major metabolites: Application of the technique to measure first-pass clearance of PGE1 in the pulmonary and cerebral circulations of the anesthetized dog

A technique for the separation of prostaglandin E₁ (PGE₁) metabolites from parent PGE₁ in large numbers of plasma samples is described. Radiolabeled metabolites were enzymatically synthesized from [³H]PGE₁, using either the 100,000g supernatant fraction from rabbit lung homogenate or perfused dog lung, and purified by thin-layer chromatography. Separation of pure 15-keto[³H]PGE_i, 13,14-dihydro-15-keto[³H]PGE₁ and 13,14-dihydro[³H]PGE₁ from parent [³H]PGE₁ was achieved on microcolumns of silica gel. All three metabolites were completely eluted from the column with 10 ml of 0.1% formic acid in ethyl acetate. Less than 5 per cent of applied [³H]PGE₁ appeared in the metabolite fraction. [³H]PGE₁ was subsequently eluted from the column with 1% formic acid in ethyl acetate. Identical results were obtained with radiolabeled metabolites and parent PGE₁ extracted from dog blood. Application of this method to indicator-dilution experiments designed to measure removal of [³H]PGE₁ in dog lung and brain is reported. Pulmonary extraction and metabolism of [³H]PGE₁ at the peak of indicator-dilution outflow curves were 84.1 ± 2.2 and 41.8 ± 9.3 per cent (mean ± S.D., N = 20) respectively. Brain extraction from duplicate runs in one animal averaged 22 per cent with less than 5 per cent metabolism of [³H]PGE₁. These estimates agree well with previously reported data and thereby support the reliability and usefulness of this new method.     

Benzodiazepine induced state-dependent learning: A correlative of abuse potential?

Using a two-way avoidance procedure, the two major benzodiadepines, diazepam and chlordiazepoxide, were tested for state-dependent learning effects in rats. Results showed that compared to saline control animals, subjects receiving diazepam or chlordiazepoxide during extinction displayed significantly more rapid extinction but a significantly greater amount of spontaneous recovery of the conditioned avoidance response. No significant dose-response differences were found. These findings are consistent with Overton's hypothesis that drug state discriminability and state-dependent learning parallel a compound's abuse potential. Drug state discriminability vs state-dependent learning is discussed and a potential relationship between state dependent properties and physical dependence is suggested.     

Correlation between polydrug abuse and alpha density

Alpha density was computed on six polydrug users and six nonusers to determine whether drug use may be related to basal levels of alpha density and/or efficiency of alpha conditioning. Alpha density was computed during four consecutive daily sessions, with each session consisting of one no-feedback and three alpha contingent, auditory feedback periods. The polydrug users were found to have greater alpha density than the nonusers under both the feedback and no-feedback conditions. Alpha contingent feedback did not increase alpha density in either group. It is suggested that psychophysiological parameters may be important variables in determining the etiology of drug use.     

Glucose metabolism of oxidatively stressed human red blood cells incubated in plasma or medium containing physiologic concentrations of lactate,pyruvate and ascorbate

Red cells suspended in either defined medium or buffered plasma were oxidatively stressed by incubation in the presence of 1, 4-naphthoquinone-2-sulfonate at concentrations which caused less than 50% methemoglobin accumulation, stimulation of the hexose monophosphate shunt to less than 15% of capacity, and about a 30% increase in flux through glycolysis. Normal plasma concentrations of lactate and pyruvate in either defined medium or buffered plasma allowed increased contribution of reducing equivalents from glycolysis in response to oxidative stress. Increased utilization of reducing equivalents by the red cell was observed as increased accumulation of pyruvate, whereas accumulation of lactate represented storage of reducing equivalents. Exogenous lactate or pyruvate did not serve as a net electron source or sink since the total content in red cell suspensions of both lactate and pyruvate was increased during exposure to oxidative stress. If exogenous lactate had been used as a net source of reducing equivalents, the lactate concentration would have decreased during incubation of red cell suspensions. Plasma ascorbate or other constituents did not alter the qualitative response of glycolysis to oxidative stress (decreased lactate accumulation, increased pyruvate accumulation, and increased total flux through glycolysis), but plasma constituents did raise significantly the dose of oxidant agent required to elicit a given quantitative response. At levels of oxidative stress likely to be encountered in vivo, glycolysis and the hexose monophosphate shunt may be equal in importance as aerobic/antioxidant pathways.     

Studies on the mechanism of potentiation of the acute toxicity of 2,4-D n-butyl ester and 2′,5-dichloro-4′-nitrosalicylanilide in rainbow trout by carbaryl

The acute toxicity of several xenobiotic substances to rainbow trout including 2,4-D n-butyl ester (2,4-DB) and 2′,5-dichloro-4′-nitrosalicylanilide (Bayer 73) has been shown to be potentiated by carbaryl. The effect seen with carbaryl was also observed when arecoline, a direct acting cholinergic agent was substituted for carbaryl. The potentiating effect of both carbaryl and arecoline was abolished by atropine. Carbaryl, at a concentration which potentiates 2,4-DB and Bayer 73, was found to increase the blood and total body concentrations of these compounds during exposures in water. Since carbaryl did not decrease the rate of elimination of 2,4-DB and Bayer 73 from preloaded fish, it appears that carbaryl may increase the acute toxicity of these compounds by increasing their uptake from water by trout. In addition to blocking the increase in acute toxicity seen with carbaryl and arecoline, atropine was also capable of blocking the increase in uptake of 2,4-DB and Bayer 73 produced by carbaryl and arecoline. It was concluded that carbaryl increases the acute toxicity of 2,4-DB and Bayer 73 by increasing the uptake of these compounds from water, possibly by effects on gill blood flow or permeability.     

Plasma protein binding and interaction studies with diflunisal,a new salicylate analgesic

The binding of diflunisal to human serum albumin and normal human plasma has been studied by equilibrium dialysis at 37°, pH 4. The plasma protein binding data were analysed according to a Scatchard model with two independent classes of binding sites. The number of binding sites and the corresponding association constants have been estimated by nonlinear least-squares regression analysis: N₁ = 2.1, k₁ = 5.28 × 10⁵M^?1, N₂ = 7.7 and K₂ = 0.17 × 10⁵M^?1. At a difluni concentration of 50 μg/ml on average 99.83 per cent of the drug was bound to plasma proteins. The in vitro plasma protein binding of diflunisal was impaired by salicylic acid and phenprocoumon, while diflunisal itself was displaced from its primary binding sites in plasma by salicylic acid and bilirubin. Tolbutamide had no effect on the binding of diflunisal to plasma proteins.